阅读说明：本技术 使用工程化的肝细胞生长因子二聚体片段进行眼部治疗的方法 (Methods of ocular treatment using engineered dimeric fragments of hepatocyte growth factor ) 是由 J·R·科克伦 于 2020-04-27 设计创作，主要内容包括：本发明提供了HGF多肽变体,包括二聚体,所述HGF多肽变体用于治疗,特别是治疗眼部疾病和眼部疾患。(The present invention provides HGF polypeptide variants, including dimers, that are useful in therapy, particularly in the treatment of ocular diseases and disorders.)

1. A method of treating and/or preventing an ocular disease and/or ocular disorder in a subject in need thereof, the method comprising administering to the subject a human hepatocyte growth factor (hgf) variant as described herein.

2. The method of claim 1, wherein the ocular disease and/or disorder is a Persistent Corneal Epithelial Defect (PCED).

3. The method of claim 1 or 2, wherein the hgf variant comprises at least one member selected from the group consisting of: amino acid substitutions, amino acid deletions, amino acid additions, and combinations thereof.

4. The method of claims 1-3, wherein the hgf variant is a homodimer.

5. The method of claims 1-4, wherein the hHGF variant is an NK1 homodimer, the NK1 homodimer comprising a homodimer of a sequence according to any one of SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, and/or SEQ ID No. 30 and an optional linker.

6. The method of claims 1-5, wherein said hHGF variant comprises at least one amino acid substitution at position 62, 127, 137, 170, or 193.

7. The method of claims 1-6, wherein said hgf variant comprises at least one amino acid substitution selected from the group consisting of: K62E, N127D/A/K/R, K137R, K170E, and N193D.

8. The method of claims 1-7, wherein the hgf variant comprises the amino acid substitutions K62E, N127D/a/K/R, K137R, K170E, and N193D.

9. The method of claims 1-8, wherein the hgf variant is an antagonist of Met.

10. The method of claims 1-9, wherein the hgf variant is an agonist of Met.

11. The method of claims 1-10, wherein the hgf variant is conjugated to a member selected from the group consisting of: a detectable moiety, a water-soluble polymer, a water-insoluble polymer, a therapeutic moiety, a targeting moiety, and combinations thereof.

12. The method of claims 1-11, wherein said hgf variant further comprises an amino acid substitution at one or more of positions 64, 77, 95, 125, 130, 132, 142, 148, 154, and 173.

13. The method of claims 1-11, wherein the hgf variant comprises a sequence selected from the group consisting of SEQ ID No. 2 through SEQ ID No. 22 from U.S. patent No.9,556,248 provided with fig. 10).

14. The method of claims 1-11, wherein the hgf variant comprises the amino acid substitutions K62E, Q95R, I125T, N127D/a/K/R, I130V, K132N/R, K137R, K170E, Q173R, and N193D.

15. The method of claim 14, wherein said hgf variant further comprises an amino acid substitution at one or more of positions 64, 77, 142, 148, and 154.

16. The method of claims 1-11, wherein the hgf variant comprises the amino acid substitutions K62E, Q95R, K132N, K137R, K170E, Q173R, and N193D.

17. The method of claim 16, wherein said hgf variant further comprises an amino acid substitution at one or more of positions 64, 77, 125, 127, 130, 142, 148, and 154.

18. The method of claims 1-11, wherein the hgf variant comprises the amino acid substitutions K62E, Q95R, N127D/a/K/R, K132N/R, K137R, K170E, Q173R, and N193D.

19. The method of claim 18, wherein said hgf variant further comprises an amino acid substitution at one or more of positions 64, 77, 125, 130, 142, 148, and 154.

20. The method of claims 1-19, wherein the hgf variant comprises a sequence selected from the group consisting of SEQ ID No. 2 through SEQ ID No. 22.

21. The method of claims 1-19, wherein the hgf variant is NK1 homodimer, the NK1 homodimer comprising a homodimer of a sequence according to any one of SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, and/or SEQ ID NO 30 and an optional linker.

22. The method of claims 1-19, wherein the hgf variant is an NK1 homodimer, the NK1 homodimer comprising a homodimer of a sequence according to SEQ ID NO: 25.

23. The method of claims 1-4, wherein said hHGF variant is an NK1 homodimer, said NK1 homodimer comprising a homodimer of a sequence according to SEQ ID NO. 26.

Technical Field

The present invention relates to the field of polypeptide variants for ocular therapy, in particular Hepatocyte Growth Factor (HGF) variants.

Background

Human growth factors play a key role in orchestrating many complex processes such as wound healing, tissue regeneration, angiogenesis and tumor formation^1-4. Thus, there is great interest in using growth factors as protein therapeutics to accelerate wound healing and regeneration processes, or to inhibit cancer growth and angiogenesis in a variety of diseases and disorders^5-7. However, even though many recombinant growth factors have been developed as therapeutic agents, only a few candidates have been effective enough to gain clinical approval^8,9. This is due in large part to the short effective half-life of growth factors in vivo, due to their generally poor stability and rapid blood clearance⁵,¹⁰. Therapeutic growth factors must remain effective in the wound area for an extended period of time to be effective. However, growth factors may be denatured or degraded when exposed to physiological temperatures and proteases^11,12. Resistance to protease-mediated degradation may be particularly important, as proteases such as plasmin and metalloproteinases are particularly active in tissue remodeling¹³。

Hepatocyte Growth Factor (HGF), also known as Scatter Factor (SF), is a multifunctional heterodimeric protein produced primarily by mesenchymal cells and is an effector of cells expressing the Met tyrosine kinase receptor ("c-Met") (Bottaro et al (1991) SCIENCE 251: 802-371; Rubin et al (1993) BIOCHIM. BIOPHYS. ACTA 1155: 357-371). Mature HGF contains two polypeptide chains, an α chain and a β chain. Published studies indicate that the alpha chain contains the c-Met receptor binding domain of HGF.

Mature HGF contains two polypeptide chains, an α chain and a β chain. HGF mediates many cellular activities upon binding to its cognate receptor. The HGF-Met signaling pathway plays a role in liver regeneration, wound healing, nerve regeneration, angiogenesis, and malignancy. See, e.g., Cao et al (2001) PROC. NATL. ACAD. SCI. USA 98: 7443-; burgess et al (2006) CANCER RES.66: 1721-.

Dysregulation of cell signaling pathways that mediate proliferation, survival and migration are potential causes of many cancers. In particular, dysregulation and overexpression of the Met tyrosine kinase receptor are associated with poor prognosis in many human tumors, making it an attractive target for therapeutic intervention.

There are currently no FDA-approved therapies targeting Met receptors, however, some candidate molecules are in different stages of clinical trials. Thus, molecules that strongly inhibit Met receptor activation can have a significant impact on cancer therapy. Furthermore, there is very limited research to develop Met-targeted molecular imaging agents for non-invasive visualization of Met expression in vivo compared to other cancer targets. The availability of such imaging agents would aid in cancer diagnosis, staging and disease management, and in determining which patients would be good candidates for Met-targeted therapy.

With respect to the eye, despite its protective role as the dome-shaped, outermost tissue of the eye, the normally clear cornea is highly susceptible to ulceration, scarring and opacification as a result of injury or disease. In severe corneal injuries and diseases, permanent scarring and vision loss often ensues, although a number of, primarily supportive, measures are currently available.¹End-stage corneal blindness is characterized by angiogenesis and opacification of one or more of the normal stratum lucidum of the cornea, followed by edema and fibrotic scarring. Almost every blinding disorder of the ocular surface, whether infectious (e.g., severe cornea)Ulcerative or herpetic keratitis), immune-mediated (e.g., stevens-johnson syndrome), or traumatic (e.g., alkali burn), all begin with impaired healing of epithelial defects and end with an opaque vascularized cornea. Tissue-derived therapies, such as serum eye drops 1 and amniotic membrane 2, are widely used clinically, but the molecular composition and underlying mechanisms of these two therapies remain unclear. 2 in contrast, a single recombinant growth factor, such as Epidermal Growth Factor (EGF), has failed clinical trials, and 3 suggests that multifactorial intervention is required to fully support corneal wound healing.

The present invention fills this need by providing methods of using Hepatocyte Growth Factor (HGF) variants for the treatment and/or prevention of ocular diseases and/or disorders, including, for example, Persistent Corneal Epithelial Defects (PCEDs) and corneal angiogenesis.

Disclosure of Invention

The present invention provides a method of treating and/or preventing an ocular disease and/or disorder in a subject in need thereof, the method comprising administering to the subject a human hepatocyte growth factor (hgf) variant as described herein.

In some embodiments, the present invention provides an ocular treatment method comprising administering a human hepatocyte growth factor (hgf) variant. In some embodiments, the ocular therapy is for Persistent Corneal Epithelial Defects (PCED) and corneal angiogenesis.

In some embodiments, the ocular disease and/or disorder is a Persistent Corneal Epithelial Defect (PCED).

In some embodiments, the hgf variant comprises at least one member selected from the group consisting of SEQ ID NO: 8: amino acid substitutions, amino acid deletions, amino acid additions, and combinations thereof.

In some embodiments, the hgf variant is a homodimer.

In some embodiments, the hgf variant is an NK1 homodimer, said NK1 homodimer comprising a sequence according to any one of SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, and/or SEQ ID NO 30, and optionally a linker.

In some embodiments, a hgf variant comprises at least one amino acid substitution at position 62, 127, 137, 170, or 193.

In some embodiments, a hgf variant comprises at least one amino acid substitution selected from the group consisting of: K62E, N127D/A/K/R, K137R, K170E, and N193D.

In some embodiments, a hgf variant comprises the amino acid substitutions K62E, N127D/a/K/R, K137R, K170E, and N193D.

In some embodiments, a hgf variant is an antagonist of Met.

In some embodiments, a hgf variant is an agonist of Met.

In some embodiments, the hgf variant is conjugated to a member selected from the group consisting of: a detectable moiety, a water-soluble polymer, a water-insoluble polymer, a therapeutic moiety, a targeting moiety, and combinations thereof.

In some embodiments, the hgf variant further comprises an amino acid substitution at one or more of positions 64, 77, 95, 125, 130, 132, 142, 148, 154, and 173.

In some embodiments, the hgf variant comprises a sequence selected from the group consisting of SEQ ID No. 2 through SEQ ID No. 22 from U.S. patent No.9,556,248 provided in accompanying figure 10).

In some embodiments, the hgf variant comprises the amino acid substitutions K62E, Q95R, I125T, N127D/a/K/R, I130V, K132N/R, K137R, K170E, Q173R, and N193D.

In some embodiments, the hgf variant further comprises an amino acid substitution at one or more of positions 64, 77, 142, 148, and 154.

In some embodiments, the hgf variant comprises the amino acid substitutions K62E, Q95R, K132N, K137R, K170E, Q173R, and N193D.

In some embodiments, the hgf variant further comprises an amino acid substitution at one or more of positions 64, 77, 125, 127, 130, 142, 148, and 154.

In some embodiments, the hgf variant comprises the amino acid substitutions K62E, Q95R, N127D/a/K/R, K132N/R, K137R, K170E, Q173R, and N193D.

In some embodiments, the hgf variant further comprises an amino acid substitution at one or more of positions 64, 77, 125, 130, 142, 148, and 154.

In some embodiments, the hgf variant comprises a sequence selected from the group consisting of SEQ ID No. 2 through SEQ ID No. 22.

Drawings

FIG. 1 schematic representation of NK1 homodimers and recombinant HGF.

FIG. 2 is a schematic diagram of the Met pathway.

Fig. 3 provides data showing CEC (corneal epithelial cells) treated with eNK1 and HGF with increased wound closure rate.

Fig. 4 provides data showing that eNK1 and HGF treated cells have increased cell proliferation.

Fig. 5 provides data showing MTT proliferation assays demonstrating CEC treated with eNK1 and rHGF for 48 hours has increased metabolic activity compared to untreated cells (p < 0.05).

Fig. 6A-6 b provide examples of IgG1, IgG2, IgG3, and IgG4 sequences.

FIG. 7 Structure of HGF domain. N: an N-terminal PAN module; k: a Kringle domain; SPH: a serine protease homology domain. The black arrows indicate the cleavage sites for cleaving HGF into its two-chain active form. The alpha and beta chains are linked by disulfide bonds. The N-terminal and first Kringle domain constitute the NK1 fragment of HGF.

FIG. 8. overview of NK1 engineering strategy. In the first round of directed evolution (M1), the library was screened for functional binding to Met; for the second round (M2), the library was screened in parallel for enhanced affinity or enhanced stability; for the third round (M3), the M2 product was shuffled and simultaneously screened for improved affinity and stability.

Figure 9. corneal wound healing study after alkali burn comparing corneal wound at (a) t 0 hours, (B) 24 hours after treatment with MSC secreting group in HA/CS gel delivery vehicle, (C) 24 hours after saline instillation alone. It has been demonstrated in preliminary work that (D) hgf alone, developed at the Cochran laboratory (PDB structure shown in inset), can also accelerate wound closure time in rat alkali burned corneas in vivo.

FIG. 10 provides the variant hepatocyte growth factor sequences SEQ ID NO 2 through SEQ ID NO 22 from U.S. Pat. No.9,556,248, which is incorporated herein by reference in its entirety.

Detailed Description

I.Brief introduction to the drawings

The Met receptor is expressed from a single gene product and is proteolytically processed into a 50kD alpha chain and a 140kD beta chain. The first 212 residues of the alpha and beta chains constitute the Sema domain, a complex 7-leaf beta propeller fold. The remainder of the Met β chain includes a cysteine-rich domain, four immunoglobulin domains, an intracellular kinase domain, and a C-terminal tail. Upon ligand binding and dimerization, Met is cross-phosphorylated at Tyr-1234 and Tyr-1235 of the intracellular kinase domain. This activity leads to further phosphorylation of Tyr-1349 and Tyr-1356 at the C-terminal end of Met, which is the multi-substrate docking site for adaptor proteins and signal transducers Shc, Grb2, Gab1, PI-3 kinase and PLC- γ.

HGF exhibits an overall domain structure similar to that of a coagulation factor such as plasminogen; it is expressed as a single-chain inactive precursor that must be cleaved into its functionally active form by enzymes such as HGF activators, matriptase, hepsin, factor XIIa and factor XIIa. Both single-chain proHGF and cleaved double-chain HGF bind Met with high affinity, but only double-chain HGF is able to induce Met activation. The HGF alpha chain consists of one N-terminal hairpin-containing domain (PAN module; globular domain) followed by four Kringle domains (fig. 1). HGF β chain consists of serine protease homeodomain, but lacks catalytic activity due to the lack of key residues in the catalytic triad.

Several HGF fragments have been reported; NK1 and NK2 are naturally occurring splice variants of HGF, and NK4 was originally discovered by digestion of HGF with pancreatic elastase. These fragments consist of the N-terminal and first Kringle (NK1), first and second kringles (NK2), or first to fourth Kringle (NK4) domains. NK1 and NK2 were initially reported as Met antagonists, but were later identified as acting as weak Met agonists. In contrast, NK4 retained strong binding to Met (400-600pM), but did not induce Met activation, thereby acting as a competitive HGF antagonist. NK1 appears to constitute the smallest functional unit of HGF, as it binds and activates Met, albeit much weaker than full-length HGF.

The invention provides methods of using HGF variants as described herein in methods of treatment associated with ocular diseases and conditions.

II.Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic and nucleic acid chemistry, and hybridization are those well known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al, M)_OLECULAR C_LONING:A L_ABORATORY M_ANUALSecond edition, (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference), which are conventional in the art and various general references are provided throughout this document. The nomenclature used herein and the analytical and synthetic organic chemistry laboratory procedures described below are those well known and commonly employed in the art. Chemical synthesis and chemical analysis are performed using standard techniques or modifications thereof.

The terms "M2.1" and "M2.2" refer to variants of seq.id No. 2 having the following substitutions, respectively: (i) K62E, N127D, K137R, K170E, N193D; and (ii) K62E, Q95R, N127D, K132N, K137R, K170E, Q173R, N193D.

In some embodiments, the term "eNK 1" refers to SEQ ID NO. 23.

As used herein, "NK 1" consists of the N-terminus and the first Kringle domain of hepatocyte growth factor. (see, e.g., Cioce, JBC,271:13110-13115 (1996)) breakpoints in polypeptides of the invention include amino acids 28-210 of human hepatocyte growth factor isoform 1(Genbank accession ID NP-000592). Others use break points of 31-210 and 32-210. Alternative human hepatocyte growth factor isoforms, isoform 3(Genbank accession ID NP _00101932.1) are identical to human hgf (hgf) isoform 1, except for the deletion of 5 amino acids in the first Kringle domain. Both hgf isoform 1 and isoform 3 efficiently activate the Met receptor, and NK1 proteins derived from hgf isoform 1 or isoform 3 also both bind to and activate the Met receptor. The breakpoints 28-205, 31-205 and 32-205 of NK1 based on isoform 3 variants will be identical to the breakpoints 28-210, 31-210 and 32-210 of NK1 based on isoform 1 variants, the only difference being the deletion of 5 amino acids from the first Kringle domain (K1).

In some embodiments, NK1 refers to SEQ ID NO:25, as set forth below, which includes an N-terminal cysteine for dimerization:

in some embodiments, NK1 refers to SEQ ID NO 26, as set forth below, which includes an N-terminal cysteine for dimerization:

in some embodiments, NK1 refers to Uniprot reference P14210:

in some embodiments, NK1 refers to Uniprot reference P14210-2:

in some embodiments, NK1 refers to Uniprot reference P14210-4:

in some embodiments, NK1 refers to protein database number (PDB)1NK1_ a or 1NK1_ B, each of which is listed by the following sequence:

the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof, in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260:2605-2608 (1985); and Rossolini et al, mol.cell.Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene. Furthermore, as used herein, a nucleic acid encoding a polypeptide variant of the present invention is defined to include a nucleic acid sequence complementary to the nucleic acid sequence.

The term "gene" refers to a segment of DNA involved in the production of a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

The term "isolated" when applied to a nucleic acid or protein means that the nucleic acid or protein is substantially free of other cellular components with which it is associated in nature. Although it may be in the form of a dry or aqueous solution, it is preferably in a homogeneous state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein, which is the major material present in the preparation, is substantially purified. Specifically, the isolated gene is separated from the open reading frames flanking the gene and encoding proteins other than the gene of interest. The term "purified" means that the nucleic acid or protein produces a substantially single band (band) in the electrophoresis gel. Specifically, this means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. The isolated nucleic acid may be a component of an expression vector.

Generally, the isolated polypeptides of the invention have a purity level preferably expressed as a range. The lower limit of the purity range for a polypeptide is about 60%, about 70%, or about 80%, and the upper limit of the purity range is about 70%, about 80%, about 90%, about 95%, or greater than about 95%. When polypeptides are greater than about 90% pure, their purity is also preferably expressed as a range. The lower limit of the purity range is about 90%, about 92%, about 94%, about 96%, or about 98%. The upper limit of the purity range is about 92%, about 94%, about 96%, about 98%, or about 100% purity.

Purity is determined by any art-recognized analytical method (e.g., band intensity on silver stained gel, polyacrylamide gel electrophoresis, HPLC, mass spectrometry, or the like).

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that have been later modified, such as hydroxyproline, γ -carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon, a carboxyl group, an amino group, and an R group bound to a hydrogen, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "amino acid mimetics" refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

"hydrophilic amino acid" refers to an amino acid that exhibits a hydrophobicity of less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al, 1984, J.mol.biol.179: 125-142. Genetically encoded hydrophilic amino acids include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K), and Arg (R).

"acidic amino acid" refers to a hydrophilic amino acid having a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of hydrogen ions. Genetically encoded acidic amino acids include Glu (E) and Asp (D).

"basic amino acid" refers to a hydrophilic amino acid having a side chain pK value greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ions. Genetically encoded basic amino acids include His (H), Arg (R), and Lys (K).

"polar amino acid" refers to a hydrophilic amino acid that has an uncharged side chain at physiological pH, but has at least one bond in which a pair of electrons shared in common by two atoms is held more tightly by one of the atoms. Genetically encoded polar amino acids include Asn (N), Gln (Q), Ser (S), and Thr (T).

"hydrophobic amino acid" refers to an amino acid that exhibits a hydrophobicity greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg,1984, J.mol.biol.179: 125-142. Exemplary hydrophobic amino acids include ile (i), phe (f), val (v), leu (l), trp (w), met (m), ala (a), gly (g), tyr (y), pro (p), and proline analogs.

"aromatic amino acid" refers to a hydrophobic amino acid having a side chain with at least one aromatic or heteroaromatic ring. The aromatic or heteroaromatic ring may contain one or more substituents, such as-OH, -SH, -CN, -F, -Cl, -Br, -I, -NO₂、-NO、-NH₂、-NHR、-NRR、-C(O)R、-C(O)OH、-C(O)OR、-C(O)NH₂-C (O) NHR, -C (O) NRR, and the like, wherein each R is independently (C)₁-C₆) Alkyl, substituted (C)₁-C₆) Alkyl, (C)₁-C₆) Alkenyl, substituted (C)₁-C₆) Alkenyl, (C)₁-C₆) Alkynyl, substituted (C)₁-C₆) Alkynyl, (C)_1-C₂₁) Aryl, substituted (C)₅-C₂₀) Aryl group, (C)₆-C₂₆) Alkylaryl, substituted (C)₆-C₂₆) Alkylaryl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 membered alkylheteroaryl, or substituted 6-26 membered alkylheteroaryl. Genetically encoded aromatic amino acids include phe (f), tyr (y), and trp (w).

"non-polar amino acid" refers to a hydrophobic amino acid having a side chain that is uncharged at physiological pH and has a bond in which a pair of electrons shared in common by two atoms is generally held equally by each of the two atoms (i.e., the side chain is not polar). Genetically encoded nonpolar amino acids include Leu (L), Val (V), Ile (I), Met (M), Gly (G), and Ala (A).

"aliphatic amino acid" refers to a hydrophobic amino acid having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include Ala (A), Val (V), Leu (L), and Ile (I).

Amino acid residue cys (c) is unusual in that it can form a disulfide bridge with other cys (c) residues or other sulfonyl-containing amino acids. The ability of the cys (c) residue (and other amino acids with-SH containing side chains) to be present in the peptide in reduced free-SH or oxidized disulfide bridge form affects whether the cys (c) residue contributes net hydrophobic or hydrophilic character to the peptide. Although cys (c) exhibits a hydrophobicity of 0.29 according to the Eisenberg normalized consensus scale (Eisenberg,1984, supra), it is understood that, despite the general classification defined above, cys (c) is classified as a polar hydrophilic amino acid for the purposes of the present invention.

The term "linker" refers to an amino acid polypeptide spacer region that covalently links two or more polypeptides. The linker may be 1-15 amino acid residues. Preferably, the linker is a single cysteine residue. The linker may also have the amino acid sequence SEQ ID NO 24KESCAKKQRQH MDS.

As will be appreciated by those skilled in the art, the categories defined above are not mutually exclusive. Thus, amino acids having side chains exhibiting two or more physicochemical properties may be included in a plurality of classes. For example, an amino acid side chain having an aromatic moiety further substituted with a polar substituent such as tyr (y) may exhibit both aromatic hydrophobic and polar or hydrophilic properties, and thus may be included in both the aromatic and polar classes. The appropriate classification of any amino acid will be apparent to those skilled in the art, especially in light of the detailed disclosure provided herein.

When certain amino acid residues, referred to as "helix-disrupting" amino acids, are contained at internal positions within the helix, they have a tendency to disrupt the alpha helical structure. Amino acid residues exhibiting such helix breaking properties are well known in the art (see, e.g., Chou and Fasman, Ann. Rev. biochem.47:251-276) and include Pro (P), Gly (G), and potentially all D-amino acids (when included in an L-peptide; conversely, when included in a D-peptide, L-amino acids disrupt helix structure) as well as proline analogs. Although these helix-disrupting amino acid residues fall into the above-defined categories, with the exception of gly (g) (discussed below), these residues should not be used to substitute amino acid residues at internal positions within the helix-they should only be used to substitute 1-3 amino acid residues at the N-terminus and/or C-terminus of the peptide.

Although the above categories have been exemplified with respect to genetically encoded amino acids, amino acid substitutions need not be, and in certain embodiments are preferably not limited to, genetically encoded amino acids. In fact, many preferred peptides of formula (I) contain non-genetically encoded amino acids. Thus, in addition to naturally occurring genetically encoded amino acids, the amino acid residues in the core peptide of formula (I) may be substituted with naturally occurring non-coding amino acids and synthetic amino acids.

Some common amino acids that provide useful substitutions for the core peptide of formula (I) include, but are not limited to, beta-alanine (beta-Ala) and other omega-amino acids (such as 3-aminopropionic acid, 2, 3-diaminopropionic acid (Dpr), 4-aminobutyric acid, etc.); α -aminoisobutyric acid (Aib); epsilon-aminocaproic acid (Aha); delta-aminovaleric acid (Ava); n-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline (Cit); tert-butylalanine (t-BuA); tert-butylglycine (t-BuG); n-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal); 4-chlorophenylalanine (Phe (4-Cl)); 2-fluorophenylalanine (Phe (2-F)); 3-fluorophenylalanine (Phe (3-F)); 4-fluorophenylalanine (Phe (4-F)); penicillamine (Pen); 1/2/3/4-tetrahydroisoquinoline-3-carboxylic acid (Tic); beta-2-thienylalanine (Thi); methionine Sulfoxide (MSO); homoarginine (hArg); n-acetyl lysine (AcLys); 2, 4-diaminobutyric acid (Dbu); 2, 3-diaminobutyric acid (Dab); p-aminophenylalanine (Phe (pNH 2)); n-methylvaline (MeVal); homocysteine (hCys), homophenylalanine (hPhe), and homoserine (hSer); hydroxyproline (Hyp), high proline (hPro), N-methylated amino acids, and peptoids (N-substituted glycines). In addition, in some embodiments, the amino acid proline in the core peptide of formula (I) is substituted with proline analogs including, but not limited to, azetidine-2-carboxylate (A2C), L-thiazolidine-4-carboxylic acid, cis-4-hydroxy-L-proline (CHP), 3, 4-dehydroproline, thioproline, and isohexide nicotinic acid (Inp).

Amino acids may be referred to herein by their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-IUB biochemical nomenclature commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

With respect to amino acid sequences, those skilled in the art will recognize that a single substitution, deletion, or addition to a nucleic acid, peptide, polypeptide, or protein sequence that alters, adds, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the invention.

The following eight groups each contain amino acids that are conservative substitutions for one another:

1) alanine (a), glycine (G);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V);

6) phenylalanine (F), tyrosine (Y), tryptophan (W);

7) serine (S), threonine (T); and

8) cysteine (C), methionine (M)

(see, e.g., Creighton, Proteins (1984)).

Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into account one or more of the foregoing characteristics are well known to those of skill in the art and include, but are not limited to (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Accordingly, embodiments of the present disclosure contemplate functional or biological equivalents of the polypeptides or proteins described above. In particular, embodiments of the invention provide variants having about 50%, 60%, 70%, 80%, 90% and 95% sequence identity to a parent polypeptide. In various embodiments, the invention provides variants having this level of identity to a portion of a parent polypeptide sequence, e.g., a wild-type growth factor, including, e.g., wild-type HGF (SEQ ID NO: 8). In various embodiments, the variant has at least about 95%, 96%, 97%, 98% or 99% sequence identity to the parent polypeptide or a portion of the parent polypeptide sequence, e.g., a wild-type growth factor, including, e.g., wild-type HGF (SEQ ID NO:8), as defined herein.

"conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each alanine position specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variant of the nucleic acid. One skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each such sequence.

As known in the art, "identity" is a relationship between two or more polypeptide or protein sequences, as determined by comparing the sequences. In the art, "identity" also refers to the degree of sequence relatedness between polypeptides or proteins, as determined by the match between strings of such sequences. "identity" can be readily calculated by known bioinformatic methods.

"peptide" refers to a polymer in which the monomers are amino acids and are joined together by amide bonds. The size of the peptides of the invention may vary, for example, from two amino acids to hundreds or thousands of amino acids. Larger peptides (e.g., at least 10, at least 20, at least 30, or at least 50 amino acid residues) may alternatively be referred to as "polypeptides" or "proteins. In addition, unnatural amino acids such as beta-alanine, phenylglycine, homoarginine, and homophenylalanine are also included. Non-genetically encoded amino acids may also be used in the present invention. In addition, amino acids that have been modified to include reactive groups, glycosylation sequences, polymers, therapeutic moieties, biomolecules, and the like, can also be used in the present invention. All amino acids used in the present invention may be d-isomer or l-isomer. The L-isomer is generally preferred. In addition, other peptidomimetics may also be used in the present invention. As used herein, "peptide" or "polypeptide" refers to both glycosylated and non-glycosylated peptides or "polypeptides". Also included are polypeptides that are incompletely glycosylated by the system of expressed polypeptides. For a general review, see Spatola, A.F., Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, B.Weinstein, Marcel Dekker, New York, page 267 (1983).

In the present application, amino acid residues are numbered (usually in the superscript) according to their relative position with respect to the N-terminal amino acid (e.g., the N-terminal methionine) of the polypeptide, which is numbered "1". The N-terminal amino acid may be methionine (M), numbered "1". If the N-terminus of the polypeptide is initially free of methionine, the number associated with each amino acid residue can be readily adjusted to reflect the deletion of the N-terminal methionine. It is understood that the N-terminus of an exemplary polypeptide may begin with or without a methionine. Therefore, in the case of adding an amino acid linker to the N-terminus of the wild-type polypeptide, the first linker amino acid adjacent to the N-terminal amino acid is the number-1 or the like. For example, if the linker has the amino acid sequence KESCAKKQRQHMDS (SEQ ID NO:2) wherein the S residue is contiguous with the N-terminal amino acid of the wild-type polypeptide, the N-most linker amino acid K will be-14 and the C-most linker amino acid S will be-1. In this way, the numbering of the amino acids in the wild-type polypeptide and the wild-type polypeptide bound to the linker is retained.

The term "parent polypeptide" refers to a wild-type polypeptide, and the amino acid sequence or nucleotide sequence of the wild-type polypeptide is part of a publicly accessible protein database (e.g., EMBL nucleotide sequence database, NCBI Entrez, ExPasy, protein database, etc.).

The term "mutant polypeptide" or "polypeptide variant" or "mutein" or "variant polypeptide" refers to a form of a polypeptide in which its amino acid sequence differs from that of its corresponding wild-type (parental) form, naturally occurring form, or any other parental form. The mutant polypeptide may contain one or more mutations, e.g., substitutions, insertions, deletions, and the like, which result in the mutant polypeptide.

The term "corresponds to a parent polypeptide" (or grammatical variations of the term) is used to describe a polypeptide of the invention in which the amino acid sequence of the polypeptide differs from the amino acid sequence of the corresponding parent polypeptide only by the presence of at least one amino acid change. Typically, the amino acid sequences of the variant and parent polypeptides exhibit a high percentage of identity. In one example, "corresponding to a parent polypeptide" refers to a variant polypeptide having an amino acid sequence that is at least about 50% identical, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% identical to the amino acid sequence of the parent polypeptide. In another example, the nucleic acid sequence encoding the variant polypeptide has at least about 50% identity, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% identity to the nucleic acid sequence encoding the parent polypeptide. In some embodiments, the parent polypeptide corresponds to the NK1 homodimer of SEQ ID NO. 23. In some embodiments, the parent polypeptide corresponds to the NK1 homodimer of SEQ ID NO. 25. In some embodiments, the parent polypeptide corresponds to the NK1 homodimer of SEQ ID NO. 26. In some embodiments, the parent polypeptide corresponds to the NK1 homodimer of SEQ ID NO. 27. In some embodiments, the parent polypeptide corresponds to the NK1 homodimer of SEQ ID NO 28. In some embodiments, the parent polypeptide corresponds to the NK1 homodimer of SEQ ID NO. 29. In some embodiments, the parent polypeptide corresponds to the NK1 homodimer of SEQ ID NO 30.

The term "introducing (or adding, etc.) variation into" (or grammatical variations thereof) a parent polypeptide, "or" modifying a parent polypeptide "to include variation (or grammatical variations thereof) does not necessarily mean that the parent polypeptide is the physical starting material for such transformation, but rather that the parent polypeptide provides the amino acid sequence for guidance in preparing the variant polypeptide. In one example, "introducing a variation into a parent polypeptide" refers to modifying a gene of the parent polypeptide by appropriate mutation to produce a nucleotide sequence encoding the variant polypeptide. In another example, "introducing variation into a parent polypeptide" refers to theoretically designing the resulting polypeptide using the parent polypeptide sequence as a guide. The designed polypeptide may then be produced by chemical or other means.

The term "library" refers to a collection of different polypeptides, each corresponding to a common parent polypeptide. Each polypeptide species in the library is referred to as a member of the library. Preferably, the library of the invention represents a collection of polypeptides in sufficient quantity and diversity to provide a population from which to identify the lead polypeptide. The library comprises at least two different polypeptides. In one embodiment, the library comprises from about 2 to about 100,000,000 members. In another embodiment, the library comprises from about 10,000 to about 100,000,000 members. In another embodiment, the library comprises from about 100,000 to about 100,000,000 members. In yet another embodiment, the library comprises from about 1,000,000 to about 100,000,000 members. In another embodiment, the library comprises from about 10,000,000 to about 100,000,000 members. In yet another embodiment, the library comprises more than 100 members.

The members of the library may be part of a mixture, or may be separated from each other. In one example, the members of the library are part of a mixture, which optionally includes other components. For example, at least two polypeptides are present in a volume of cell culture fluid. In another example, the members of the library are each expressed separately, and may optionally be isolated. The isolated polypeptide may optionally be contained in a multi-well container in which each well contains a different type of polypeptide. In another example, the members of the library are each expressed as fusions to yeast or bacterial cells or phage or viral particles.

As used herein, the term "polymeric modifying group" is a modifying group comprising at least one polymeric moiety (polymer). Polymeric modifying groups added to polypeptides may alter the properties of such polypeptides, such as their bioavailability, biological activity or their half-life in vivo. Exemplary polymers include water soluble polymers and water insoluble polymers. The polymer modifying group may be linear or branched and may comprise one or more independently selected polymeric moieties such as poly (alkylene glycol) s and derivatives thereof. In one example, the polymer is non-naturally occurring. In an exemplary embodiment, the polymer modifying group includes water soluble polymers such as poly (ethylene glycol) and its derivatives (PEG, m-PEG), poly (propylene glycol) and its derivatives (PPG, m-PPG), and the like. In a preferred embodiment, the molecular weight of the poly (ethylene glycol) or poly (propylene glycol) is substantially homogeneously dispersed. In one embodiment, the polymeric modifying group is not a naturally occurring polysaccharide.

As used herein, the term "targeting moiety" refers to a substance that will selectively localize in a particular tissue or region of the body. Localization is mediated by specific recognition of molecular determinants, molecular size of the targeting agent or conjugate, ionic interactions, hydrophobic interactions, and the like. Other mechanisms for targeting an agent to a particular tissue or region are known to those skilled in the art. Exemplary targeting moieties include antibodies, antibody fragments, transferrin, HS-glycoprotein, coagulation factors, serum proteins, beta-glycoprotein, G-CSF, GM-CSF, M-CSF, EPO, and the like.

As used herein, the term "Fc fusion protein" is intended to include proteins, particularly therapeutic proteins, which comprise an immunoglobulin-derived portion, which portion will be referred to herein as the "Fc portion"; and a portion derived from a second protein that is not an immunoglobulin, which portion will be referred to herein as a "therapeutic portion", regardless of whether the disease is intended to be treated.

As used herein, "therapeutic moiety" refers to any agent useful in therapy, including, but not limited to, antibiotics, anti-inflammatory agents, antineoplastic agents, cytotoxins, and radioactive agents. "therapeutic moiety" includes prodrugs of biologically active agents, constructs in which more than one therapeutic moiety is conjugated to a carrier, such as multivalent agents.

Therapeutic moieties also include proteins and constructs comprising proteins.

As used herein, "antineoplastic agent" refers to any agent useful against cancer, including.

As used herein, "cytotoxic or cytotoxic agent" refers to any agent that is harmful to a cell. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emidine, mitomycin, etoposide, tigoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. Other toxins include, for example, ricin, CC-1065 and analogs, ducamycin. Other toxins also include diphtheria toxin and snake venom (e.g., cobra venom).

As used herein, "radioactive agent" includes any radioisotope effective in diagnosing or destroying a tumor. Examples include, but are not limited to, indium-111, cobalt-60, fluorine-18, copper-64, copper-67, lutetium-177, or technetium-99 m. Additionally, naturally occurring radioactive elements, such as uranium, radium and thorium, which generally represent a mixture of radioisotopes, are suitable examples of radioactive agents. The metal ion is typically chelated to the organic chelating moiety. The radioactive agent or radionuclide may be a component of the imaging agent.

For optical imaging applications, the near-infrared dye can also be conjugated using standard chemical methods. "near infrared" refers to radiation in a portion of the electromagnetic spectrum adjacent to the portion associated with visible light, for example, from about 0.7 μm to about 1 μm. Near infrared dyes may include, for example, cyanine or indocyanine green derivatives, such as Cy5.5. The infrared dyes may also include phosphoramidite dyes, for example800(Biosciecnes)。

Many useful chelating groups, crown ethers, cryptands, and the like are known in the art and can be incorporated into the compounds of the invention (e.g., EDTA, DTPA, DOTA, NTA, HDTA, and the like, as well as their phosphonate analogs such as DTPP, EDTP, HDTP, NTP, and the like). See, e.g., Pitt et al, "The Design of chemical Agents for The Treatment of Iron Overload," INORGANIC CHEMISTRY IN BIOLOGY AND M_EDICINE(ii) a Martell eds; american Chemical Society, Washington, D.C.,1980, p.279-312; lindoy, T_HEC_{HEMISTRY OF} M_ACROCYCLIC L_IGAND COMPLEXES；Cambridge University Press,Cambridge,1989；Dugas,BIOORGANIC C_HEMISTRY(ii) a Springer-Verlag, New York,1989, and references contained therein. In addition, a variety of approaches are available to those skilled in the art that allow for the attachment of chelators, crown ethers, and cyclodextrins to other molecules. See, e.g., Meares et al, "Properties of In Vivo chemistry-Tagged Proteins and polypeptides," M_{ODIFICATION OF} P_ROTEINS:F_OOD,N_{UTRITIONAL,AND} P_{HARMACOLOGICAL} A_SPECTS(ii) a Feeney et al, American Chemical Society, Washington, D.C.,1982, pp.370-387; kasina et al, Bioconjugate chem.,9: 108-; song et al, Bioconjugate chem.,8:249-255 (1997). These metal binding agents can be used to bind metal ions that are detectable in an imaging modality.

As used herein, "pharmaceutically acceptable carrier" includes any material that retains the activity of the conjugate when combined with the conjugate and is non-reactive with the immune system of the subject. "pharmaceutically acceptable carriers" include solids and liquids, such as vehicles, diluents, and solvents. Examples include, but are not limited to, any standard pharmaceutical carrier such as phosphate buffered saline solution, water, emulsions such as oil/water emulsions, and various types of wetting agents. Other carriers may also include sterile solutions, tablets, including coated tablets, and capsules. Typically, such carriers comprise excipients such as starch, milk, sugar, certain types of clays, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavoring and color additives or other ingredients. Compositions comprising such carriers are formulated by well-known conventional methods.

As used herein, "administering" means orally administering, administering as a suppository, topically contacting, intravenously, intraperitoneally, intramuscularly, intrathecally, intralesionally, or subcutaneously administering to a subject, administering by inhalation, or implanting a sustained release device, e.g., a mini osmotic pump. Administration is by any route, including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal), particularly by inhalation. Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Furthermore, where the injection is used to treat a tumor (e.g., induce apoptosis), the tumor may be administered directly to and/or into tissue surrounding the tumor. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

The term "ameliorating" refers to any indication of success in the treatment of a pathology or disorder, including any objective or subjective parameter, such as reduction, alleviation or relief of symptoms or improvement in the physical or mental well-being of a patient. The amelioration of symptoms can be based on objective or subjective parameters; including the results of physical examination and/or psychiatric evaluation.

The term "therapy" refers to "treatment" of a disease or disorder, including preventing the disease or disorder from occurring in a subject (e.g., a human) susceptible to the disease but not yet experiencing or exhibiting symptoms of the disease (prophylactic treatment), inhibiting the disease (delaying or arresting its development), providing relief from the symptoms or side effects of the disease (including palliative treatment), and relieving the disease (resulting in regression of the disease).

The term "effective amount" or "an amount effective for … …" or "therapeutically effective amount" or any grammatical equivalent term means an amount sufficient to effect treatment of a disease when administered to an animal or human to treat the disease. An effective amount may also refer to an amount necessary to elicit a cellular response including, for example, apoptosis, cell cycle initiation, and/or signal transduction.

The term "pharmaceutically acceptable salt" includes salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When the compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral forms of such compounds with a sufficient amount of the desired base, neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonium or magnesium salts, or similar salts. When the compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydroiodic, or phosphorous acids and the like, as well as salts derived from relatively nontoxic organic acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids, such as arginine salts and the like, and salts of organic acids, such as glucuronic acid or galacturonic acid and the like (see, e.g., Berge et al, Journal of Pharmaceutical Science,66:1-19 (1977)). Certain specific compounds of the invention contain both basic and acidic functionalities that allow the compounds to be converted into base addition salts or acid addition salts.

The neutral form of the compound is preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties (such as solubility in polar solvents), but otherwise the salts are equivalent to the parent form of the compound for purposes of this invention.

The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compound may be used with a compound such as tritium (f)³H) Iodine-125 (¹²⁵I) Or carbon-14 (¹⁴C) Is radiolabeled with the radioisotope of (a). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

As used herein, "reactive functional group" refers to groups including, but not limited to: olefins, acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazos, nitro groups, nitriles, thiols, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acids, isonitriles, amidines, imides, imidoesters, nitrones, hydroxylamines, oximes, hydroxamic acids, thiohydroxamic acids, and the like,Allene, orthoester, sulfite, enamine, acetylenic amine, urea, pseudourea, semicarbazide, carbodiimide, carbamate, imine, azide, azo compound, azoxy compound, and nitroso compound. Reactive functional groups also include those used to prepare bioconjugates, for example, N-hydroxysuccinimide esters, maleimides, and the like. Methods for preparing each of these functional groups are well known in the art, and their use or modification for a particular purpose is within the ability of those skilled in the art (see, e.g., Sandler and Karo, eds., O_RGANIC F_UNCTIONAL G_ROUPP_REPARATIONS,Academic Press,San Diego,1989)。

III.HFG variants

In some embodiments, the variant is a proteolytically stable variant as compared to the wild-type growth factor. In an exemplary embodiment, the variant exhibits increased proteolytic stability compared to the wild type. In some embodiments, the variant is any variant of a wild-type growth factor. In some embodiments, the variant is an antagonist of a growth factor receptor to which the wild-type growth factor binds.

The present invention provides a hgf polypeptide comprising at least one amino acid not found in a parent hgf polypeptide (wild type) in at least one position of the amino acid. The present invention encompasses variants of all isoforms of hgf, including but not limited to isoforms 1 and 3. Isoform 3(NCBI accession No. NP _001010932) includes the following five amino acid deletions (SFLPS) underlined in seq.id No. 8 (isoform 1).

In an exemplary embodiment, the present invention provides HGF variants of seq.id.no. 9 having at least one amino acid substitution.

In an exemplary embodiment, the variant is an isolated variant. Furthermore, in various embodiments, the variant exhibits at least one desired characteristic not present in the polypeptide of the invention. Exemplary features include, but are not limited to, increased affinity for the Met receptor, increased thermostability, increased or decreased conformational flexibility, and increased agonistic or antagonistic activity to the Met receptor. As will be appreciated by those skilled in the art, the variant may exhibit any combination of two or more of these improved characteristics.

In an exemplary embodiment, the polypeptide variant is an antagonist of Met receptor. In various embodiments, the variant is an agonist of the Met receptor.

In an exemplary embodiment, the present invention provides an hgf polypeptide variant having a sequence that is a member selected from seq.id No. 9.

An exemplary parent polypeptide is wild-type HGF isoform 1(HGF NCBI accession number NP-000592) (SEQ. ID NO:8)

In seq.id No. 8, the signal peptide comprises amino acids 1-31. The N-terminal domain comprises amino acids 39-122. The Kringle 1 domain comprises amino acids 126-207; kringle 2 comprises amino acids 208-289; kringle 3 comprises amino acids 302-384; kringle 4 comprises amino acids 388-470. The serine protease-like domain comprises 495-719.

In exemplary embodiments, the variants of the invention have at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96%, 97%, 98%, or 99% sequence identity to the parent polypeptide. In various embodiments, the variants of the invention have at least about 99.2%, at least about 99.4%, at least about 99.6%, or at least about 99.8% sequence identity to the parent polypeptide.

In an exemplary embodiment, the mutation positions of seq.id No.9 include one or more of 62, 64, 77, 95, 125, 127, 130, 132, 137, 142, 148, 154, 170, 173, and 193. As will be appreciated by those skilled in the art, any combination of these positions may be mutated. In various embodiments, analogous positions of isoform 3 are mutated.

In an exemplary embodiment, the amino acid of the parent polypeptide is changed from K to a member selected from E, N and R. In an exemplary embodiment, the amino acid in the parent polypeptide is changed from Q to R. In an exemplary embodiment, the amino acid in the parent polypeptide is changed from I to a member selected from T and V. In an exemplary embodiment, the amino acid of the parent polypeptide is changed from N to D. In some embodiments, D may revert back to the N of the parent polypeptide.

In various embodiments, the amino acid at position 42 is F or C. In various embodiments, the amino acid at position 62 is changed from K to E found in the wild-type parent polypeptide. In various embodiments, at position 64 is V or A. In various embodiments, at position 77 is N or S. In various embodiments, the amino acid at position 95 is Q or R. In various embodiments, the amino acid at position 125 is changed from I to T found in the wild-type parent polypeptide. In various embodiments, the amino acid at position 127 can be D, N, K, R or a. In various embodiments, the amino acid at position 130 is changed from I to V. In various embodiments, the amino acid at position 132 is changed from K to N or R. In various embodiments, the amino acid at position 137 is K or R. In various embodiments, the amino acid at position 154 is S or a. In various embodiments, the amino acid at position 170 is K or E. In various embodiments, the amino acid at position 173 is Q or R. In various embodiments, the amino acid at position 193 is N or D. In various embodiments, the amino acid at position 42 is F or C. In various embodiments, the amino acid at position 96 is C or R. As will be appreciated by those skilled in the art, any combination of these variations, as well as any combination of those listed in the following table, may be present in a polypeptide variant of the invention.

In some embodiments, an HGF variant comprises K62E, N127D, K170E, and N193E as compared to wild-type HGF (SEQ ID NO: 9). In some embodiments, an HGF variant comprises K62E, Q95R, N127D, K132N, K170E, Q173R, and N193E as compared to wild-type HGF (SEQ ID NO: 9).

In some embodiments, an HGF variant comprises a consensus sequence having the following specific amino acids at the listed positions, as compared to wild-type HGF (SEQ ID No. 9): K62E, Q95R, I125T, N127D, I130V, K132N, K137R, K170E, Q173R, and N193E.

Table 1, table 2, and table 3 show exemplary mutations of HGF variants described herein.

Table 1.

TABLE 2 respective sequence mutations of NK1 mutants isolated from the third round of directed evolution. SEQ ID NO 8 is wild type; the only differences from the wild type sequence are shown in SEQ ID NO 9; blank area indicates retention of wild-type hgf residue.

bp: number of base pair mutations

AA: number of amino acid mutations

TABLE 3 respective sequence mutations of NK1 mutants isolated from the third round of directed evolution. SEQ ID NO 9 is wild type; the only difference from the wild type sequence is shown in SEQ ID NO 9.

bp: number of base pair mutations

AA: number of amino acid mutations

a.Conjugates

The invention provides conjugates of the variants of the invention with one or more conjugation partners. Exemplary conjugation partners include polymers, targeting agents, therapeutic agents, cytotoxic agents, chelating agents, and detectable agents. One skilled in the art will recognize that there is overlap between these non-limiting agent classes.

The conjugation partner or "modifying group" may be any moiety that can be conjugated. Exemplary modifying groups are discussed below. Modification groups may be selected based on their ability to alter a characteristic (e.g., a biological or physicochemical characteristic) of a given polypeptide. Exemplary polypeptide properties that can be altered by the use of modifying groups include, but are not limited to, pharmacokinetics, pharmacodynamics, metabolic stability, biodistribution, water solubility, lipophilicity, tissue targeting ability, and therapeutic activity profile. The modifying groups can be used to modify polypeptides used in diagnostic applications or in vitro bioassay systems.

In some embodiments, a growth factor variant comprising an HGF variant, e.g., as described herein, is combined with an Fc portion. The Fc portion may be derived from a human or animal immunoglobulin (Ig), which is preferably an IgG. The IgG may be IgG1, IgG2, IgG3, or IgG4 (see, e.g., fig. 34). It is also preferred that the Fc portion is derived from the heavy chain of an immunoglobulin, preferably IgG. More preferably, the Fc portion comprises a portion of an immunoglobulin heavy chain constant region, such as a domain. The Ig constant region preferably comprises at least one Ig constant domain selected from any one of the hinges, CH2, CH3 domains, or any combination thereof. In some embodiments, the Fc portion comprises at least CH2 and CH3 domains. It is further preferred that the Fc portion comprises an IgG hinge region, CH2 and CH3 domains.

Table 4: exemplary IgG sequences:

the Fc domain of the IgG1 subclass is commonly used as the Fc moiety because IgG1 has the longest serum half-life of any serum protein. The long serum half-life may be a desirable protein profile for animal research and potential human therapeutic use. In addition, the IgG1 subclass has the strongest ability to perform antibody-mediated effector functions.

The major effector function most useful in fusion proteins is the ability of the IgG1 antibody to mediate antibody-dependent cellular cytotoxicity. On the other hand, for fusion proteins that act primarily as antagonists, this may be an undesirable function. Several specific amino acid residues important for antibody constant region-mediated activity in the IgG1 subclass have been identified. Thus, the inclusion or exclusion of these specific amino acids allows the inclusion or exclusion of specific immunoglobulin constant region-mediated activities.

According to the invention, the Fc portion may also be modified to modulate effector function. For example, if the Fc portion is derived from IgG1, the following Fc mutations can be introduced according to the EU index position (Kabat et al, 1991): T250Q/M428L; M252Y/S254T/T256E + H433K/N434F; E233P/L234V/L235A/A Δ 236+ A327G/A330S/P331S; E333A; K322A.

A further Fc mutation may be, for example, a substitution at the EU index position selected from 330, 331, 234 or 235 or a combination thereof. In the context of the present invention, an amino acid substitution at EU index position 297 in the CH2 domain may also be introduced into the Fc portion, thereby eliminating potential N-linked carbohydrate attachment sites. The cysteine residue at EU index position 220 may also be substituted.

The Fc fusion protein of the present invention may be a monomer or a dimer. The Fc fusion protein may also be a "pseudo-dimer" containing dimeric Fc portions (e.g., dimers of two disulfide-bridged hinge-CH 2-CH3 constructs) of which only one is fused to a therapeutic moiety.

The Fc fusion protein may be a heterodimer comprising two different therapeutic moieties, or a homodimer comprising two copies of a single therapeutic moiety.

In some embodiments, the in vivo half-life of growth factor variants described herein, including, for example, HGF variants, can be extended with polyethylene glycol (PEG) moieties. Chemical modification (pegylation) of a polypeptide with PEG increases the molecular size of the polypeptide and typically decreases the accessibility of surfaces and functional groups, each of which depends on the number and size of PEG moieties attached to the polypeptide. Generally, such modifications result in improved plasma half-life and proteolytic stability, as well as reduced immunogenicity and liver uptake (Chaffee et al, J.Clin.invest.89:1643-1651 (1992); Pyratak et al, Res.Commun.Chem.Pathol Pharmacol.29:113-127 (1980)). For example, it is reported that pegylation of interleukin 2 increases its antitumor efficacy in vivo (Katre et al, Proc. Natl. Acad. Sci. USA.84: 1487-42-1394 (1987)), and that pegylation of F (ab') 2 derived from monoclonal antibody A7 improves its tumor localization (Kitamura et al, biochem. Biophys. Res. Commun.28:1387-1394 (1990)). Thus, in another embodiment, the in vivo half-life of a polypeptide derivatized with a PEG moiety by the methods of the invention is increased relative to the in vivo half-life of the non-derivatized parent polypeptide.

The increase in vivo half-life of a polypeptide is best expressed as a percentage increase range relative to the parent polypeptide. The lower limit of the range of percent increase is about 40%, about 60%, about 80%, about 100%, about 150%, or about 200%. The upper end of the range is about 60%, about 80%, about 100%, about 150%, or greater than about 250%.

Many water-soluble polymers are known to those skilled in the art and can be used in the practice of the present invention. The term water-soluble polymer encompasses materials such as: sugars (e.g., dextran, amylose, hyaluronic acid, poly (sialic acid), heparinoids, heparin, etc.); poly (amino acids), such as poly (aspartic acid) and poly (glutamic acid); a nucleic acid; synthetic polymers (e.g., poly (acrylic acid), poly (ether) s such as poly (ethylene glycol); peptides, proteins, etc.. the invention may be practiced with any water-soluble polymer, the only limitation being that the polymer must contain a point at which the remaining conjugate can be attached see, for example, Harris, Macronol. chem. Phys. C25:325-373 (1985); Scouten, Methods in Enzymology 135:30-65 (1987); Wong et al, Enzyme Microb. technology.14: 1992-874 (Delgado et al, clinical Reviews in Therapeutic Drug Systems 9:249 (1992); Zalipsky, Bioconjugate chem.6:150-165 (1995); and Bhadra et al, pharmaceutical 57:5-29 (2002)).

In another embodiment, the modified sugar comprises a water insoluble polymer, rather than a water soluble polymer, similar to those discussed above. The conjugates of the invention may also include one or more water-insoluble polymers. This embodiment of the invention is illustrated by using the conjugate as a vehicle with which the therapeutic polypeptide is delivered in a controlled manner. Polymeric drug delivery systems are known in the art. See, e.g., Dunn et al, POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series, Vol 469, American Chemical Society, Washington, D.C. 1991. Those skilled in the art will appreciate that essentially any known drug delivery system is suitable for use in the conjugates of the invention.

Representative water insoluble polymers include, but are not limited to, polyphosphazines, poly (vinyl alcohols), polyamides, polycarbonates, polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinyl pyrrolidones, polyglycolides, polysiloxanes, polyurethanes, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), poly (octadecyl acrylate) polyethylene, polypropylene, poly (ethylene glycol), poly (ethylene glycol), poly (ethylene glycol), poly (ethylene glycol), poly (ethylene glycol), poly (ethylene glycol), poly (ethylene glycol), poly (ethylene glycol), poly (ethylene glycol), poly (ethylene glycol, and poly (ethylene glycol), poly (ethylene oxide), poly (ethylene terephthalate), poly (vinyl acetate), polyvinyl chloride, polystyrene, polyvinylpyrrolidone, pluronics, and polyvinylphenol, and copolymers thereof.

Representative biodegradable polymers for use in the conjugates of the present invention include, but are not limited to, polylactides, polyglycolides and their copolymers, poly (ethylene terephthalate), poly (butyric acid), poly (valeric acid), poly (lactide-co-caprolactone), poly (lactide-co-glycolide), polyanhydrides, polyorthoesters, blends and copolymers thereof. Gel-forming compositions are particularly useful, such as those comprising collagen, pluronic, and the like.

Exemplary absorbable polymers include, for example, synthetically produced absorbable block copolymers of poly (alpha-hydroxy-carboxylic acid)/poly (oxyethylene) (see, Cohn et al, U.S. patent No. 4,826,945). These copolymers are not crosslinked and are water soluble so that the body can excrete the degraded block copolymer composition. See, Youtes et al, J biomed.Mater.Res.21:1301- & 1316 (1987); and Cohn et al, J biomed.Mater.Res.22:993-1009 (1988).

Polymers that are components of hydrogels may also be useful in the present invention. Hydrogels are polymeric materials that are capable of absorbing relatively large amounts of water. Examples of hydrogel-forming compounds include, but are not limited to, polyacrylic acid, sodium carboxymethylcellulose, polyvinyl alcohol, polyvinyl pyrrolidine, gelatin, carrageenan and other polysaccharides, Hydroxyethylmethacrylate (HEMA), derivatives thereof, and the like. Stable, biodegradable and bioabsorbable hydrogels can be produced. Further, the hydrogel composition may comprise subunits that exhibit one or more of these properties.

In another embodiment, the gel is a thermoreversible gel. Presently preferred are thermally reversible gels comprising components such as: pluronic, collagen, gelatin, hyaluronic acid, polysaccharides, polyurethane hydrogels, polyurethane-urea hydrogels, and combinations thereof.

In another exemplary embodiment, the conjugates of the invention comprise a component of a liposome. Liposomes can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 to Eppstein et al, issued 6/11 1985. For example, liposomal formulations can be prepared by: suitable lipids (e.g., stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachidoyl phosphatidyl choline, and cholesterol) are dissolved in an inorganic solvent, which is then evaporated away, leaving a dry lipid film on the surface of the container. An aqueous solution of the active compound or a pharmaceutically acceptable salt thereof is then introduced into the container. The container was then swirled by hand to release the lipid material from the sides of the container and disperse the lipid aggregates, thereby forming a liposome suspension.

The invention also provides conjugates similar to the conjugates described above, wherein the polypeptide is conjugated to a therapeutic moiety, a diagnostic moiety, a targeting moiety, a toxin moiety, and the like. Each of the above moieties may be a small molecule, a natural polymer (e.g., a polypeptide), or a synthetic polymer.

In various embodiments, the variant is conjugated to a component of a matrix for tissue regeneration. Exemplary matrices are known in the art and are within the ability of one skilled in the art to select and modify suitable matrices with growth factor variants of the invention, including, for example, HGF variants. The growth factor variants of the invention, including, for example, HGF variants, are commonly used in regenerative medicine applications, including, for example, regeneration of ocular, hepatic, muscular, neural, and cardiac tissue.

In some embodiments, the invention provides conjugates that selectively localize in a particular tissue due to the presence of a targeting agent that is a component of the conjugate. In exemplary embodiments, the targeting agent is a protein. Exemplary proteins include transferrin (brain, blood pool), HS glycoprotein (bone, brain, blood pool), antibody (brain, tissue with antibody-specific antigen, blood pool), coagulation factor V-XII (damaged tissue, clot, cancer, blood pool), serum proteins (e.g., alpha-acid glycoprotein, fetuin, alpha-fetuin (brain, blood pool), beta 2-glycoprotein (liver, atherosclerotic plaque, brain, blood pool)), G-CSF, GM-CSF, M-CSF, and EPO (immune stimulation, cancer, blood pool, overproduction of erythrocytes, neuroprotection), albumin (extended half-life), IL-2, and IFN-alpha.

In another embodiment, the invention provides a method of treating a disease or disorder comprising administering to a subject a growth factor variant of the invention (including, e.g., an HGF variant) and a therapeutic moietyThe conjugate of (1). Therapeutic moieties useful in the practice of the present invention include drugs from a variety of drug classes with a variety of pharmacological activities. Methods of conjugating therapeutic and diagnostic agents to various other substances are well known to those skilled in the art. See, e.g., Hermanson, B_IOCONJUGATE T_ECHNIQ_UESAcademic Press, San Diego, 1996; and Dunn et al, P_OLYMERIC D_RUGS A_ND D_RUG D_ELIVERY S_YSTEMSACS Symposium Series, volume 469, American Chemical Society, Washington, d.c. 1991.

Classes of useful therapeutic moieties include, for example, antineoplastic agents (e.g., antiandrogens (e.g., leuprolide or flutamide), cytocidal agents (e.g., doxorubicin, paclitaxel, cyclophosphamide, busulfan, cisplatin, beta-2-interferon), antiestrogens (e.g., tamoxifen), antimetabolites (e.g., fluorouracil, methotrexate, mercaptopurine, thioguanine).

The therapeutic moiety can also be a hormone (e.g., medroxyprogesterone, estradiol, leuprolide, megestrol, octreotide, or somatostatin); endocrine regulating drugs (e.g., contraceptives (e.g., norethindrone, ethinyl estradiol, norethindrone, mestranol, desogestrel, medroxyprogesterone)). Used in various embodiments of the present invention are conjugates with estrogens (e.g., diethylstilbestrol), glucocorticoids (e.g., triamcinolone, betamethasone, etc.), and progestins (such as norethindrone, levonorgestrel); a thyroid agent (e.g., liothyronine or levothyroxine) or an antithyroid agent (e.g., methimazole); conjugates of anti-hyperprolactinemic drugs (e.g., cabergoline); hormonal inhibitors (e.g., danazol or goserelin), oxytocin (e.g., methylergonovine or oxytocin) and prostaglandins such as misoprostol, alprostadil or dinoprostone may also be used.

Other useful modifying groups include immunomodulatory drugs (e.g., antihistamines, mast cell stabilizers such as lodoxylamine and/or cromolyn), steroids (e.g., triamcinolone, beclomethazone, cortisone, dexamethasone, prednisolone, methylprednisolone, beclomethasone or clobetasol), histamine H2 antagonists (e.g., famotidine, cimetidine, ranitidine), immunosuppressive agents (e.g., azathioprine, cyclosporine), and the like. Groups with anti-inflammatory activity may also be used, such as sulindac, etodolac, ketoprofen and ketorolac. Other drugs for use in conjunction with the present invention will be apparent to those skilled in the art.

In some embodiments, the conjugate is formed by a reaction between a reactive amino acid and a reactive conjugation partner of the reactive amino acid. Both the reactive amino acid and the reactive conjugation partner comprise one or more reactive functional groups within their framework. One of the two binding substances may include a "leaving group" (or activating group) which refers to those moieties (e.g., amino acid moieties bearing a sulfhydryl group) that are easily replaced in an enzymatically mediated nucleophilic substitution reaction, or are replaced in a chemical reaction with a nucleophilic reaction partner. It is within the ability of the skilled person to select a suitable leaving group for each type of reaction. Many activated sugars are known in the art. See, e.g., Vocadlo et al, C_ARBOHYDRATE C_{HEMISTRY AND} B_IOLOGYVol.2, Ernst et al, Wiley-VCH Verlag, Weinheim, Germany, 2000; kodama et al Tetrahedron Lett.34:6419 (1993); loughed et al, J.biol.chem.274:37717 (1999)).

In various embodiments, the amino acid substitution is a naturally occurring variant of HGF (the variant or a variant) that is a site for attachment of a conjugation partner (e.g., a side chain amino acid, such as cysteine, lysine, serine, etc.).

Reactive groups and classes of reactions useful in practicing the invention are generally bioconjugate chemistryThose well known in the art. The class of advantageous reactions with reactive sugar moieties currently available are those that proceed under relatively mild conditions. These include, but are not limited to, nucleophilic substitutions (e.g., reaction of amines and alcohols with acid halides, activated esters), electrophilic substitutions (e.g., enamine reactions), and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reactions, Diels-Alder additions). These and other useful reactions are described in, for example, March, A_DVANCED O_RGANIC C_HEMISTRY3 rd edition, John Wiley&Sons, New York, 1985; hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al, M_{ODIFICATION OF} P_ROTEINS(ii) a Advances in Chemistry Series, volume 198, American Chemical Society, Washington, D.C., 1982.

b.Reactive functional group

Reactive functional groups useful on reactive amino acids or reactive conjugation partners include, but are not limited to:

(a) carboxyl groups and their various derivatives, including but not limited to N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acid halides, acylimidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl, and aromatic esters;

(b) hydroxyl groups, which can be converted to, for example, esters, ethers, aldehydes, and the like;

(c) haloalkyl, wherein the halo group can be subsequently replaced by a nucleophilic group such as an amine, carboxylate anion, thiolate anion, carbanion, or alkoxide ion, resulting in covalent attachment of a new group at the functional group of the halogen atom;

(d) dienophile groups, which are capable of participating in Diels-Alder reactions, such as maleimido groups;

(e) an aldehyde or keto group, such that subsequent derivatization may occur by formation of a carbonyl derivative, such as an imine, hydrazone, semicarbazone, or oxime, or by a mechanism such as grignard addition or alkyllithium addition;

(f) a sulfonyl halide group for subsequent reaction with an amine, e.g., to form a sulfonamide;

(g) thiol groups, which can be converted, for example, to disulfides or reacted with acid halides;

(h) amine or thiol groups, which may be acylated, alkylated or oxidized, for example;

(i) olefins, which may undergo, for example, cycloaddition, acylation, Michael addition, and the like; and

(j) epoxides, which can be reacted with, for example, amines and hydroxyl compounds.

The reactive functional groups may be selected such that they do not participate in or interfere with the reactions necessary to assemble the reactive sugar core or modifying group. Alternatively, the reactive functional group may be protected from reaction by the presence of a protecting group. One skilled in the art understands how to protect a particular functional group so that it does not interfere with a selected set of reaction conditions. For examples of useful protecting groups, see, e.g., Greene et al, P_ROTECTIVE G_{ROUPS IN} O_RGANIC S_YNTHESIS,John Wiley&Sons,New York,1991。

The moiety linking the polypeptide and the conjugation partner may also be a cross-linking moiety, for example a zero or higher order cross-linking moiety (for a review of cross-linking reagents and cross-linking procedures, see: Wold, F., meth.enzymol.25:623-651, 1972; Weetall, H.H. and Cooney, D.A., E._{NZYMES AS} D_RUGS(Holcenberg and Roberts eds.) pp 395-442, Wiley, New York, 1981; ji, T.H., meth.enzymol.91:580-609, 1983; mattson et al, mol.biol.Rep.17:167-183,1993, all of which are incorporated herein by reference). Preferred crosslinking reagents are derived from various zero-length, homobifunctional and heterobifunctional crosslinking reagents. The zero-length crosslinking reagent comprises direct conjugation of two intrinsic chemical groups without the introduction of an external material. Agents that catalyze disulfide bond formation belong to this class. Another example is a reagent that induces condensation of carboxyl and primary amino groups to form amide bonds, such as carbodiimide, ethyl chloroformate, wood wadde reagent K (2-ethyl-5-phenylisoxazolium-3' -sulfonate) and carbonyldiimidazole. In addition to these chemical reagents, transglutaminase (glutamyl-peptide gamma-transglutaminase; EC 2.3.2.13) can be usedIs a zero length crosslinking reagent. This enzyme catalyzes an acyl transfer reaction at the carboxamide group of a protein-bound glutamine residue, typically with a primary amino group as substrate. Preferred homobifunctional and heterobifunctional reagents contain two identical or two different sites, respectively, which may be reactive towards amino, sulfhydryl, guanidino, indole or non-specific groups.

Exemplary conjugation partners attached to a polypeptide of the invention include, but are not limited to, PEG derivatives (e.g., alkyl-PEG, acyl-alkyl-PEG, alkyl-acyl-PEG, carbamoyl-PEG, aryl-PEG), PPG derivatives (e.g., alkyl-PPG, acyl-alkyl-PPG, alkyl-acyl-PPG, carbamoyl-PPG, aryl-PPG), therapeutic moieties, diagnostic moieties, mannose-6-phosphate, heparin, heparinoids, Sle_xMannose, mannose-6-phosphate, sialyl-lewis X, FGF, VFGF, protein, chondroitin, keratin, dermatan, albumin, integrin, antennal oligosaccharide, peptide, and the like.

In addition to covalent attachment, growth factor variants of the invention, including, for example, HGF variants, can be attached to the surface of a biomaterial by non-covalent interactions. Non-covalent protein incorporation can be accomplished, for example, by encapsulation or absorption. Attachment of the polypeptides of the invention to the biomaterial may be mediated by heparin. In some embodiments, the polypeptides of the invention are attached to heparin-alginate polymers and alginates, as described in Harada et al, J.Clin.Invest. (1994)94: 623-; laham et al, Circulation (1999)1865-1871 and the references cited therein. In other embodiments, the polypeptides of the invention are attached to collagen-based biomaterials.

c.Imaging agent

An exemplary conjugate of the invention is an imaging agent comprising a variant of the invention and a detectable moiety that is detectable under an imaging modality. There is an urgent need for molecular imaging probes that will specifically target Met receptors in living subjects and allow for non-invasive characterization of tumors for use in patient-specific cancer therapy and disease management. The ability to detect Met expressing tumors by non-invasive imaging can also be an indicator of the risk of metastasis.

Exemplary imaging modalities in which the conjugates of the invention may be used include, but are not limited to, Positron Emission Tomography (PET), wherein the variants of the invention are labeled with a positron emitting isotope. Typical isotopes include¹¹C、¹³N、¹⁵O、¹⁸F、⁶⁴Cu、⁶²Cu、¹²⁴I、⁷⁶Br、⁸²Rb and⁶⁸ga, wherein¹⁸F is the most clinically used isotope. The variants may also be incorporated into ultrasound, magnetic resonance, X-ray, CT, gamma scintigraphy and fluorescence imaging agents. Additional detectable moieties and imaging methods are set forth in the methods section below.

In an exemplary embodiment, the conjugation partner is linked to the polypeptide variant of the invention through a linkage that is cleaved under selected conditions. Exemplary conditions include, but are not limited to, a selected pH (e.g., stomach, intestine, intracellular vacuole), the presence of active enzymes (e.g., esterases, reductases, oxidases), light, heat, and the like. Many cleavable groups are known in the art. See, e.g., Jung et al, biochem, Biophys, acta,761:152-162 (1983); joshi et al, J.biol.chem.,265: 14518-; zarling et al, J.Immunol.,124:913-920 (1980); bouizar et al, Eur.J.biochem.,155:141-147 (1986); park et al, J.biol.chem.,261:205-210 (1986); browning et al, J.Immunol.,143:1859-1867 (1989).

IV.Pharmaceutical composition

The growth factor variants of the invention, including, for example, HGF variants and conjugates thereof, have a wide range of pharmaceutical applications.

Thus, in another aspect, the invention provides a pharmaceutical composition comprising at least one polypeptide or polypeptide conjugate of the invention, and a pharmaceutically acceptable diluent, carrier, vehicle, additive or combination thereof. The pharmaceutical compositions of the present invention are suitable for use in a variety of drug delivery systems. Suitable formulations for use in the present invention are presented in Remington's Pharmaceutical Sciences, machine Publishing Company, Philadelphia, Pa., 17 th edition (1985). For a brief review of methods for drug delivery, see Langer, Science 249: 1527) -1533 (1990).

The pharmaceutical composition may be formulated for any suitable mode of administration, including, for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, fat, wax or buffer. For oral administration, any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, and magnesium carbonate may be employed. Biodegradable matrices, such as microspheres (e.g., polylactate polyglycolide), may also be used as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. nos. 4,897,268 and 5,075,109.

Typically, the pharmaceutical composition is administered subcutaneously or parenterally (e.g., intravenously). Accordingly, the present invention provides compositions for parenteral administration comprising a compound dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g., water, buffered water, saline, PBS and the like. The composition may also comprise detergents, such as tween 20 and tween 80; stabilizers such as mannitol, sorbitol, sucrose and trehalose; and preservatives such as EDTA and m-cresol. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like.

These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solution may be packaged as is or in lyophilized form, with the lyophilized preparation being compounded with a sterile aqueous carrier prior to administration. The pH of the preparation will generally be between 3 and 11, more preferably from 5 to 9, most preferably from 7 to 8.

In some embodiments, the glycopeptides of the present invention may be incorporated into liposomes formed from standard vesicle-forming lipids. There are a variety of methods available for preparing liposomes, as described, for example, in Szoka et al, ann, rev, biophysis, bioeng.9:467(1980), U.S. patent nos. 4,235,871, 4,501,728, and 4,837,028. The use of a variety of targeting agents (e.g., sialylated galactosides of the present invention) to achieve liposomal targeting is well known in the art (see, e.g., U.S. patent nos. 4,957,773 and 4,603,044).

Standard methods for coupling targeting agents to liposomes can be used. These methods generally involve incorporating into the liposomes: a lipid component (e.g., phosphatidylethanolamine), which can be activated to attach a targeting agent, or derivatized lipophilic compound (e.g., a lipid-derivatized glycopeptide of the present invention).

The targeting mechanism typically requires that the targeting agent be placed on the surface of the liposome in such a way that the target moiety is available to interact with a target, such as a cell surface receptor. The carbohydrates of the invention may be attached to a lipid molecule prior to formation of liposomes using methods known to those skilled in the art (e.g., alkylation or acylation of the hydroxyl groups present on the carbohydrate with long chain alkyl halides or with fatty acids, respectively).

Alternatively, liposomes can be used in such a way that the linker moiety is first incorporated into the membrane when the membrane is formed. The linker moiety must have a lipophilic moiety that must be firmly embedded and anchored in the membrane. It must also have reactive moieties that are chemically available on the aqueous surface of the liposome. The reactive moiety is selected such that it is chemically suitable to form a stable chemical bond with a targeting agent or carbohydrate that is added later. In some embodiments, the target agent may be attached directly to the linker molecule, but in most cases it is more appropriate to use a third molecule to act as a chemical bridge, linking the linker molecule in the membrane to the target agent or carbohydrate extending three-dimensionally from the surface of the vesicle.

Growth factor variants prepared by the methods of the invention, including, for example, HGF variants, may also be used as diagnostic reagents. For example, viaThe labeled compounds are useful for locating areas of inflammation or tumor metastasis in a patient suspected of having inflammation. For this purpose, can use¹²⁵I、¹⁴C or tritium labeled compounds.

V.Nucleic acids

In some embodiments, the present invention provides an isolated nucleic acid encoding a growth factor variant according to any of the embodiments set forth above, including, for example, an HGF variant. In some embodiments, the invention provides a nucleic acid complementary to the nucleic acid.

In some embodiments, the present invention provides an expression vector comprising a nucleic acid encoding a polypeptide variant according to any of the embodiments set forth above operably linked to a promoter.

VI.Method

a.Chemical synthesis

Polypeptide variants of the invention can be prepared using conventional stepwise solution or Solid Phase Synthesis (see, e.g., Chemical intermediates to the Synthesis of Peptides and Proteins, Williams et al, 1997, CRC Press, Boca Raton Florida, and references cited therein; Solid Phase Peptide Synthesis: A Practical applications, Atherton and Sheppard, 1989, IRL Press, Oxford, England, and references cited therein).

Alternatively, the peptides of the invention may be prepared by segment condensation, as described, for example, in Liu et al, 1996, Tetrahedron Lett.37(7) 933936; baca et al, 1995, J.Am.chem.Soc.117: 1881-1887; tam et al, 1995, int.j.peptide Protein Res.45: 209-216;and Kent,1992, Science 256: 221-; liu and Tam,1994, J.am.chem.Soc.116(10): 4149-4153; liu and Tam,1994, Proc. Natl. Acad. Sci. USA 91: 6584-; yamashiro and Li,1988, int.J. peptide Protein Res.31: 322-334). Segment condensation is a particularly useful method for synthesizing embodiments comprising an internal glycine residue. Can be used forOther methods for synthesizing the peptides of the invention are described in Nakagawa et al, 1985, J.Am.chem.Soc.107: 7087-.

Polypeptide variants containing N-terminal and/or C-terminal end-capping groups can be prepared using standard organic chemistry techniques. For example, methods for acylating the N-terminus of a peptide or amidating or esterifying the C-terminus of a peptide are well known in the art. Modes for making other modifications at the N-terminus and/or C-terminus will be apparent to those skilled in the art, as a mode of protecting any side chain functional groups may be necessary for attaching a terminal blocking group. Pharmaceutically acceptable salts (counterions) can be conveniently prepared by ion exchange chromatography or other methods well known in the art.

The compounds of the invention in tandem multimeric form can be conveniently synthesized by adding linkers to the peptide chain at appropriate steps of synthesis. Alternatively, helical segments may be synthesized and each segment reacted with a linker. The actual synthesis method will, of course, depend on the composition of the linker. Suitable protection schemes and chemistries are well known and will be apparent to those skilled in the art.

Compounds of the invention in the form of branched networks can be conveniently synthesized using trimeric and tetrameric resins and chemical methods described in Tam,1988, Proc.Natl.Acad.Sci.USA 85:5409-5413 and Demoor et al, 1996, Eur.J.biochem.239: 74-84. The strategies for modifying synthetic resins and for synthesizing higher or lower order branched networks, or combinations of such branched networks comprising different core peptide helical segments, are well within the capabilities of those skilled in the art of peptide chemistry and/or organic chemistry. If desired, disulfide bond formation is generally carried out in the presence of a mild oxidizing agent.

Chemical oxidants may be used or the compounds may simply be exposed to atmospheric oxygen to effect these bonds. Various methods are known in the art, including, for example, those described by Tam et al, 1979, Synthesis 955-; stewart et al, 1984, Solid Phase Peptide Synthesis, 2 nd edition, Pierce Chemical Company Rockford, IL; ahmed et al, 1975, J.biol.chem.250: 8477-8482; and those described by Pennington et al, 1991Peptides 1990164-166, Giralt and Andreu, ESCOM Leiden, The Netherlands. Additional alternatives are described by Kamber et al, 1980, Helv. Chim. acta 63: 899-. Methods performed on solid supports are described by Albericio,1985, int.J. peptide Protein Res.26: 92-97. Any of these methods can be used to form disulfide bonds in the peptides of the present invention.

VII.Acquisition of the polypeptide coding sequence

a.General recombination technique

Variations incorporating the O-linked glycosylation sequence of the invention and/or creation of a mutant polypeptide can be achieved by mutation or by complete chemical synthesis of the polypeptide, altering the amino acid sequence of the corresponding parent polypeptide. The polypeptide amino acid sequence is preferably altered by changes at the DNA level, particularly by mutating the DNA sequence encoding the polypeptide at preselected bases to create codons that will translate into the desired amino acids. DNA mutagenesis is preferably performed using methods known in the art.

The present invention relies on conventional techniques in the field of recombinant genetics. Basic documents disclosing general methods for use in the present invention include Sambrook and Russell, Molecular Cloning, a Laboratory Manual (3 rd edition, 2001); kriegler, Gene Transfer and Expression A Laboratory Manual (1990); and Ausubel et al, Current Protocols in Molecular Biology (1994).

Nucleic acid size is given in kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, the size is given in kilodaltons (kDa) or number of amino acid residues. Protein size is estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.

Commercially unavailable oligonucleotides can be chemically synthesized, for example, according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Lett.22:1859-1862(1981) using an automated synthesizer, as described in Van Devanter et al, Nucleic Acids Res.12:6159-6168 (1984). The entire gene can also be chemically synthesized. Purification of the oligonucleotides is performed using any art-recognized strategy, such as, for example, native acrylamide gel electrophoresis or anion exchange HPLC as described in Pearson and Reanier, J.Chrom.255:137-149 (1983).

The sequence of the cloned wild-type polypeptide Gene, the polynucleotide encoding the mutant polypeptide, and the synthetic oligonucleotide may be verified after cloning using a chain termination method for sequencing double-stranded templates, such as Wallace et al, Gene 16:21-26 (1981).

In an exemplary embodiment, the glycosylation sequence is added by shuffling the polynucleotide. Polynucleotides encoding candidate polypeptides may be modulated using DNA shuffling protocols. DNA shuffling is a process of recursive recombination and mutation that is performed by: the relevant gene pool is randomly fragmented and then the fragments are reassembled by a process similar to the polymerase chain reaction. See, e.g., Stemmer, proc.natl.Acad.Sci.USA 91:10747-10751 (1994); stemmer, Nature 370:389-391 (1994); and U.S. Pat. nos. 5,605,793, 5,837,458, 5,830,721, and 5,811,238.

b.Cloning and subcloning of wild-type peptide coding sequences

Various polynucleotide sequences encoding wild-type polypeptides have been identified and may be obtained from commercial suppliers, e.g., human growth hormones, such as GenBank accession nos. NM 000515, NM 002059, NM 022556, NM 022557, NM 022558, NM 022559, NM 022560, NM 022561, and NM 022562.

The rapid progress in human genome research has made possible cloning methods in which a human DNA sequence database can be searched for any gene segment that has a percentage of sequence homology to a known nucleotide sequence (e.g., a known nucleotide sequence encoding a previously identified polypeptide). Any DNA sequence so identified may then be obtained by chemical synthesis and/or Polymerase Chain Reaction (PCR) techniques, such as overlap extension. For short sequences, complete de novo synthesis may be sufficient; however, in order to obtain larger genes, it may be necessary to further isolate the full-length coding sequence from a human cDNA or genomic library using synthetic probes.

Alternatively, nucleic acid sequences encoding the polypeptides can be isolated from human cDNA or genomic DNA libraries using standard cloning techniques, such as Polymerase Chain Reaction (PCR), where homology-based primers can generally be derived from known nucleic acid sequences encoding the polypeptides. The most common techniques used for this purpose are described in standard literature, e.g., Sambrook and Russell, supra.

A cDNA library suitable for obtaining the coding sequence for the wild-type polypeptide may be commercially available or may be constructed. General methods for isolating mRNA, preparing cDNA by reverse transcription, ligating the cDNA into a recombinant vector, transfecting into a recombinant host for propagation, screening and cloning are well known (see, e.g., Gubler and Hoffman, Gene,25:263-269 (1983); Ausubel et al, supra). After obtaining an amplified segment of the nucleotide sequence by PCR, the segment can be further used as a probe to isolate the full-length polynucleotide sequence encoding the wild-type polypeptide from the cDNA library. A general description of a suitable procedure can be found in Sambrook and Russell, supra.

Similar procedures can be followed to obtain full-length sequences encoding wild-type polypeptides from a human genomic library, e.g., any of the GenBank accession numbers mentioned above. Human genomic libraries are commercially available or can be constructed according to various art-recognized methods. Typically, to construct a genomic library, DNA is first extracted from tissues in which polypeptides may be found. The DNA is then subjected to mechanical shearing or enzymatic digestion to produce fragments of about 12-20kb in length. The fragments were then separated from polynucleotide fragments of undesired size by gradient centrifugation and inserted into phage lambda vectors. These vectors and phage were packaged in vitro. Recombinant phages were analyzed by plaque hybridization as described in Benton and Davis, Science,196:180-182 (1977). Colony hybridization was performed as described by Grunstein et al, Proc.Natl.Acad.Sci.USA,72:3961-3965 (1975).

Based on sequence homology, degenerate oligonucleotides can be designed as primer sets, and PCR can be performed under appropriate conditions (see, e.g., White et al, PCR Protocols: Current Methods and Applications, 1993; Griffin and Griffin, PCR Technology, CRC Press Inc.1994) to amplify fragments of nucleotide sequences from cDNA or genomic libraries. Using the amplified segment as a probe, a full length nucleic acid encoding the wild-type polypeptide is obtained.

After obtaining a nucleic acid sequence encoding a wild-type polypeptide, the coding sequence can be subcloned into a vector, such as an expression vector, so that a recombinant wild-type polypeptide can be produced from the resulting construct. Further modifications, such as nucleotide substitutions, may then be made to the wild-type polypeptide coding sequence to alter the properties of the molecule.

c.Introduction of mutations into polypeptide sequences

The amino acid sequence of the wild-type polypeptide can be determined from the encoding polynucleotide sequence. Subsequently, the amino acid sequence can be modified to alter the glycosylation pattern of the protein by introducing additional glycosylation sequences at various positions in the amino acid sequence.

Various mutagenesis protocols have been established and described in the art. See, e.g., Zhang et al, Proc. Natl. Acad. Sci. USA,94: 4504-; and Stemmer, Nature,370:389-391 (1994). This procedure can be used alone or in combination to generate a set of variants of the nucleic acid, and thus the encoded polypeptide. Kits for mutagenesis, library construction, and other diversity generation methods are commercially available.

Mutagenesis methods for generating diversity include, for example, site-directed mutagenesis (Botstein and short, Science,229:1193-1201(1985)), mutagenesis using uracil-containing templates (Kunkel, Proc. Natl. Acad. Sci. USA,82:488-492(1985)), oligonucleotide-directed mutagenesis (Zoller and Smith, Nucl. acids Res.,10:6487-6500(1982)), phosphorothioate-modified DNA mutagenesis (Taylor et al, Nucl. acids Res.,13:8749-8764 and 8765-8787(1985)), and mutagenesis using gapped double-stranded DNA (Kramer et al, Nucl. acids Res.,12:9441-9456 (1984)).

Other methods for generating mutations include point mismatch repair (Kramer et al, Cell,38: 879-.

d.Modification of nucleic acids for preferred codon usage in host organisms

The polynucleotide sequence encoding the polypeptide variant may be further altered to conform to the preferred codon usage of a particular host. For example, the preferred codon usage of a bacterial cell strain can be used to derive polynucleotides encoding variants of the polypeptides of the invention and including codons preferred by the strain. The preferred codon usage frequency exhibited by the host cell can be calculated by averaging the preferred codon usage frequencies among a large number of genes expressed by the host cell (e.g., computational services are available from the website of Kazusa DNA Research Institute, Japan). The analysis is preferably limited to genes highly expressed by the host cell. For example, U.S. Pat. No. 5,824,864 provides codon usage frequencies of highly expressed genes exhibited by dicotyledonous and monocotyledonous plants.

When the modification is complete, the polypeptide variant coding sequence is verified by sequencing and then subcloned into a suitable expression vector for recombinant production in the same manner as the wild-type polypeptide.

VIII.Expression of mutant Polypeptides

Following sequence validation, polypeptide variants of the invention can be produced by virtue of the polynucleotide sequence encoding a polypeptide disclosed herein using conventional techniques in the field of recombinant genetics.

a.Expression system

To obtain high levels of expression of a nucleic acid encoding a mutant polypeptide of the invention, the polynucleotide encoding the mutant polypeptide is typically subcloned into an expression vector containing a strong promoter to direct transcription, a transcription/translation terminator, and a ribosome binding site for translation initiation. Suitable bacterial promoters are well known in the art and are described, for example, in Sambrook and Russell, supra, and Ausubel et al, supra. Bacterial expression systems for expressing wild-type or mutant polypeptides are available in, for example, escherichia coli (e.coli), Bacillus (Bacillus sp.), Salmonella (Salmonella), and corynebacterium (Caulobacter). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast and insect cells are well known in the art and are also commercially available. In one embodiment, the eukaryotic expression vector is an adenoviral vector, an adeno-associated vector, or a retroviral vector.

The promoter used to direct expression of the heterologous nucleic acid will depend on the particular application. The promoter is optionally positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural environment. However, as known in the art, some variation in this distance can be tolerated without loss of promoter function.

In addition to the promoter, the expression vector typically includes a transcription unit or expression cassette that contains all the additional elements required for expression of the mutant polypeptide in the host cell. Thus, a typical expression cassette contains a promoter operably linked to a nucleic acid sequence encoding the mutant polypeptide, as well as signals required for efficient polyadenylation, ribosome binding site, and translation termination of the transcript. The nucleic acid sequence encoding the polypeptide is typically linked to a cleavable signal peptide sequence to facilitate secretion of the polypeptide by the transformed cell. Such signal peptides include, inter alia, signal peptides from tissue plasminogen activator, insulin and neuronal growth factor, and juvenile hormone esterase of Heliothis virescens (Heliothis virescens). Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.

In addition to the promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence, or may be obtained from a different gene.

The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any conventional vector for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids, such as pBR 322-based plasmids, pSKF, pET23D, and fusion expression systems, such as GST and LacZ. Epitope tags may also be added to recombinant proteins to provide convenient isolation methods, such as c-myc.

Expression vectors containing regulatory elements from eukaryotic viruses are commonly used in eukaryotic expression vectors, such as SV40 vectors, papillomavirus vectors, and vectors derived from epstein-barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺、pMTO10/A⁺pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of a protein under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells.

In some exemplary embodiments, the expression vector is selected from the group consisting of pCWin1, pCWin2, pCWin2/MBP, pCWin2-MBP-SBD (pMS)₃₉) And pCWin2-MBP-MCS-SBD (pMXS)₃₉) As disclosed in a commonly owned U.S. patent application filed on 9/4/2004, which is incorporated herein by reference.

Some expression systems have markers that provide gene amplification, such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. Alternatively, high-yield expression systems not involving gene amplification are also suitable, such as baculovirus vectors in insect cells having a polynucleotide sequence encoding a mutant polypeptide under the direction of a polyhedrin promoter or other strong baculovirus promoter.

The elements typically contained in expression vectors also include replicons that function in E.coli, genes encoding antibiotic resistance to allow selection of bacteria with recombinant plasmids, and unique restriction sites in non-essential regions of the plasmid to allow insertion of eukaryotic sequences. The particular antibiotic resistance gene selected is not critical and any of the many resistance genes known in the art are suitable.

If desired, the prokaryotic sequences are optionally selected such that they do not interfere with the replication of DNA in eukaryotic cells.

When periplasmic expression of a recombinant protein (e.g., the hgh mutant of the invention) is desired, the expression vector further comprises a sequence encoding a secretion signal (e.g., an E.coli OppA (periplasmic oligopeptide-binding protein) secretion signal or a modified form thereof) that is directly linked 5' to the coding sequence of the protein to be expressed. The signal sequence directs the recombinant protein produced in the cytoplasm through the cell membrane into the periplasmic space. The expression vector may further comprise a coding sequence for signal peptidase 1, said signal peptidase 1 being capable of enzymatically cleaving the signal sequence when the recombinant protein enters the periplasmic space. A more detailed description of periplasmic production of recombinant proteins can be found, for example, in Gray et al, Gene 39:247-254(1985), U.S. Pat. Nos. 6,160,089 and 6,436,674.

As noted above, one skilled in the art will recognize that various conservative substitutions may be made for any wild-type or mutant polypeptide or its coding sequence, while still retaining the biological activity of the polypeptide. In addition, modifications of the polynucleotide coding sequence can be made to accommodate preferred codon usage in a particular expression host without altering the resulting amino acid sequence.

b.Transfection method

Bacterial, mammalian, yeast or insect cell lines expressing large amounts of the mutant polypeptides are generated using standard transfection Methods and then purified using standard techniques (see, e.g., Colley et al, J.biol. chem.264:17619-17622 (1989); Guide to Protein Purification, Methods in Enzymology, Vol. 182 (Deutscher, 1990)). Transformation of eukaryotic and prokaryotic cells is performed according to standard techniques (see, e.g., Morrison, J.Bact.132:349-351 (1977); Clark-Curtiss and Curtiss, Methods in Enzymology 101:347-362(Wu et al, 1983).

Any well-known procedure for introducing a foreign nucleotide sequence into a host cell may be used. These procedures include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors, and any other well-known method to introduce cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell (see, e.g., Sambrook and Russell, supra). It is only necessary that the particular genetic engineering method used be capable of successfully introducing at least one gene into a host cell capable of expressing the mutant polypeptide.

c.Detecting expression of mutant polypeptide in host cell

After introduction of the expression vector into a suitable host cell, the transfected cells are cultured under conditions conducive to expression of the mutant polypeptide. The cells are then screened for expression of the recombinant polypeptide and the cells expressing the recombinant polypeptide are subsequently recovered from culture using standard techniques (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Pat. No. 4,673,641; Ausubel et al, supra; and Sambrook and Russell, supra).

Several general methods for screening gene expression are well known to those skilled in the art. First, gene expression can be detected at the nucleic acid level. Various methods of specific DNA and RNA measurement using nucleic acid hybridization techniques are commonly used (e.g., Sambrook and Russell, supra). Some methods involve electrophoretic separation (e.g., Southern blotting for detecting DNA and Northern blotting for detecting RNA), but detection of DNA or RNA can also be performed without electrophoresis (e.g., by dot blotting). The presence of nucleic acid encoding a mutant polypeptide in transfected cells can also be detected by PCR or RT-PCR using sequence specific primers.

Second, gene expression can be detected at the polypeptide level. One skilled in the art routinely uses various immunoassays to measure the level of gene products, particularly using polyclonal or monoclonal Antibodies that specifically react with the mutant polypeptides of the invention (e.g., Harlow and Lane, Antibodies, A Laboratory Manual, Chapter 14, Cold Spring Harbor, 1988; Kohler and Milstein, Nature,256: 495-. Such techniques require antibody production by selecting antibodies with high specificity for the mutant polypeptide or antigenic portion thereof. Methods for producing polyclonal and monoclonal antibodies are well established and their descriptions can be found in the literature, see, e.g., Harlow and Lane, supra; kohler and Milstein, Eur.J.Immunol.,6:511-519 (1976). In the subsequent section, a more detailed description of the preparation of antibodies against the mutant polypeptides of the invention and the performance of immunoassays to detect the mutant polypeptides is provided.

IX.Purification of recombinantly produced mutant polypeptides

Once expression of the recombinant mutant polypeptide in the transfected host cell is confirmed, the host cell is cultured at an appropriate scale for the purpose of purifying the recombinant polypeptide.

a.Purification from bacteria

When the mutant polypeptides of the invention are produced recombinantly in large quantities by transformed bacteria, usually after promoter induction, these proteins may form insoluble aggregates, although expression may be constitutive. There are several protocols suitable for purifying protein inclusion bodies. For example, purification of the collectin (hereinafter referred to as inclusion bodies) usually involves extraction, isolation, and/or purification of the inclusion bodies by disrupting the bacterial cells, for example, by incubation in a buffer containing about 100-. The cell suspension may be ground using a Polytron grinder (Brinkman Instruments, Westbury, NY). Alternatively, the cells may be sonicated on ice. Alternative methods of lysing bacteria are described in Ausubel et al and Sambrook and Russell, both supra, and will be apparent to those skilled in the art.

For further description of the Purification of recombinant polypeptides from bacterial inclusion bodies, see, e.g., Patra et al, Protein Expression and Purification 18: 182-.

The recombinant protein present in the supernatant can be separated from the host protein by standard separation techniques well known to those skilled in the art.

b.Immunoassays for detecting mutant polypeptide expression

To confirm the production of recombinant mutant polypeptides, immunoassays can be used to detect the expression of the polypeptides in a sample. Immunoassays can also be used to quantify the expression levels of recombinant hormones. Antibodies against the mutant polypeptides are necessary for performing these immunoassays.

c.Production of antibodies against mutant polypeptides

Methods for generating polyclonal and Monoclonal Antibodies that specifically react with an immunogen of interest are known to those skilled in the art (see, e.g., Coligan, Current Protocols in Immunology Wiley/Greene, NY, 1991; Harlow and Lane, Antibodies: organic Manual Spring Harbor Press, NY, 1989; Stits et al (eds.) Basic and Clinical Immunology (4 th edition) Lange Medical Publications, Los Altos, CA, and references cited therein; Goding, Monoclonal Antibodies: Principles and Practice (2 nd edition) Academic Press, New York, 1986; and NY Koer and stein Nature Mil: 495: 1975). Such techniques include antibody preparation by selecting antibodies from libraries of recombinant antibodies in phage or similar vectors (see Huse et al, Science 246: 1275. sup. laid-open 1281, 1989; and Ward et al, Nature 341: 544. sup. laid-open 546, 1989).

To generate antisera containing antibodies with the desired specificity, a polypeptide of interest (e.g., a mutant polypeptide of the invention) or antigenic fragment thereof can be used to immunize a suitable animal, such as a mouse, rabbit, or primate. Standard adjuvants, such as freund's adjuvant, can be used according to standard immunization protocols. Alternatively, a synthetic antigenic peptide derived from the particular polypeptide may be conjugated to a carrier protein and then used as an immunogen.

The immune response of the animal to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the antigen of interest. When an appropriately high titer of antibodies against the antigen is obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera can then be performed to enrich for and purify antibodies specifically reactive against the antigen, see, Harlow and Lane, supra, and the general description of protein purification provided above.

Monoclonal antibodies are obtained using a variety of techniques familiar to those skilled in the art. Typically, spleen cells from animals immunized with a desired antigen are immortalized, usually by fusion with myeloma cells (see, Kohler and Milstein, Eur.J.Immunol.6:511-519, 1976). Alternative methods of immortalization include, for example, transformation with an epstein-barr virus, oncogene, or retrovirus, or other methods well known in the art. Colonies from single immortalized cells are screened for production of antibodies with the desired specificity and affinity for the antigen, and the yield of monoclonal antibodies produced by such cells can be increased by a variety of techniques, including injection into the peritoneal cavity of a vertebrate host.

In addition, monoclonal antibodies can also be recombinantly produced when identifying nucleic acid sequences encoding antibodies or binding fragments of such antibodies having the desired specificity by screening a human B cell cDNA library according to the general protocol outlined by Huse et al, supra. The general principles and methods of recombinant polypeptide production discussed above are applicable to the production of antibodies by recombinant methods.

When desired, antibodies capable of specifically recognizing a mutant polypeptide of the present invention can be tested for cross-reactivity with wild-type polypeptides and thus be distinguished from antibodies directed against wild-type proteins. For example, antisera obtained from an animal immunized with a mutant polypeptide can be passed through a column on which the wild-type polypeptide is immobilized. The antisera fraction that passed through the column only recognized the mutant polypeptide, but not the wild-type polypeptide. Similarly, monoclonal antibodies directed against mutant polypeptides that exclusively recognize only the mutant and not the wild-type polypeptide can also be screened.

Polyclonal or monoclonal antibodies that specifically recognize only the mutant polypeptide of the invention, but not the wild-type polypeptide, can be used to isolate the mutant protein from the wild-type protein, for example, by incubating the sample with the mutant peptide-specific polyclonal or monoclonal antibody immobilized on a solid support.

X.Methods of treatment and diagnosis

In various embodiments, the invention provides methods of preventing, ameliorating, or treating a disease state that can be treated by administering an HGF variant polypeptide that inhibits Met or only weakly activates Met. In these embodiments, the invention provides a method comprising administering to a subject in need thereof an amount of an HGF variant polypeptide of the invention sufficient to prevent, ameliorate or treat a disease state, particularly an ocular disease state or condition. A typical disease state is corneal wound healing. The disclosed agonist variants are useful for promoting cell growth, and in some cases, for angiogenesis and corneal wound healing.

In some embodiments, therapies using an HGF variant polypeptide are used to treat, prevent, and/or inhibit corneal epithelial cell defects.

In some embodiments, therapy with an HGF variant polypeptide is used to treat, prevent, and/or inhibit a persistent corneal epithelial defect. In some embodiments, the persistent corneal epithelial defect has an epithelial/limbal etiology. In some embodiments, a persistent corneal epithelial defect includes, but is not limited to, epithelial basement membrane disease, recurrent erosion, post-traumatic scar, sarzmann nodular degeneration, zonal keratopathy, bullous keratopathy, toxic drug eruptions, malnutrition (vitamin a deficiency), or limbal stem cell defect. In some embodiments, the persistent corneal epithelial defect is selected from the group consisting of: epithelial basement membrane disease, recurrent erosion, post-traumatic scarring, salitsmann nodular degeneration, zonal keratopathy, bullous keratopathy, toxic drug eruptions, malnutrition (vitamin a deficiency), and limbal stem cell defect.

In some embodiments, therapy with an HGF variant polypeptide is used to treat, prevent, and/or inhibit a persistent corneal epithelial defect. In some embodiments, the persistent corneal epithelial defect has an inflammatory etiology. In some embodiments, persistent corneal epithelial defects include, but are not limited to, keratoconjunctivitis sicca, ocular rosacea (ocularis), chemical/thermal injury, post-infection keratitis, autoimmune disorders, sjogren's syndrome (ii)syndrome), pemphigoid, Stevens-Johnson syndrome (Stevens-Johnson syndrome), graft-versus-host disease, marginal ulcerative keratitis, peptic ulcer, or rheumatoid arthritis. In some embodiments, the persistent corneal epithelial defect is selected from the group consisting of: keratoconjunctivitis sicca, ocular rosacea, chemical/thermal injury, post-infection keratitis, autoimmune disorders, sjogren's syndrome, pemphigoid, stevens-johnson syndrome, graft versus host disease, limbic ulcerative keratitis, silkworm erosive ulcer, and rheumatoid arthritis.

In some embodiments, therapy with an HGF variant polypeptide is used to treat, prevent, and/or inhibit a persistent corneal epithelial defect. In some embodiments, the persistent corneal epithelial defect has a neurotrophic etiology. In some embodiments, the persistent corneal epithelial defects include, but are not limited to, diabetes, herpes simplex, herpes zoster, Riley-Day syndrome, narcotic or topical non-steroidal anti-inflammatory drug (NSAID) abuse, post-radiation, or post-corneal transplant cranial nerve V injury. In some embodiments, the persistent corneal epithelial defect is selected from the group consisting of: diabetes, herpes simplex, herpes zoster, Leili-wear syndrome, narcotic or topical non-steroidal anti-inflammatory drug (NSAID) abuse, post radiation and post corneal transplantation cranial nerve V injury.

In some embodiments, therapy with an HGF variant polypeptide is used to treat, prevent, and/or inhibit a persistent corneal epithelial defect. In some embodiments, the persistent corneal epithelial defect has a mechanical etiology. In some embodiments, the persistent corneal epithelial defects include, but are not limited to, blepharoedema/ectropion, blepharoptosis insufficiency (lagophthalmos), trichiasis, blepharospasm, pseudomembranous/meibomian scarring, trachoma, or artifact of the human body. In some embodiments, the persistent corneal epithelial defect is selected from the group consisting of: eyelid inversion/eversion, blepharospasm insufficiency, trichiasis, blepharospasm, pseudomembranous/meibomian scarring, trachoma, and anthropogenic.

In some embodiments, therapy with an HGF variant polypeptide is used to treat, prevent, and/or inhibit a persistent corneal epithelial defect. In some embodiments, the persistent corneal epithelial defect has an idiopathic etiology. In some embodiments, persistent corneal epithelial defects include, but are not limited to, aniridia or corneal stromal dystrophy. In some embodiments, the persistent corneal epithelial defect is selected from the group consisting of: no dystrophy of iris or corneal stroma.

In some embodiments, therapy with an HGF variant polypeptide is used to treat, prevent, and/or inhibit a Persistent Corneal Epithelial Defect (PCED), corneal angiogenesis, acute corneal abrasion. In some embodiments, the PCED is an ocular disease equivalent to a nonhealing (e.g., diabetic) ulcer of the foot. In some embodiments, PCED occurs when the process of epithelial healing and defect closure is delayed, resulting in corneal epithelial defects that can lead to ulceration, infection, scarring, perforation and loss of vision. In some embodiments, PCED may be caused by injury, previous ophthalmic surgery, infection (e.g., previous herpes infection or severe bacterial ulcer), or ocular disease (including underlying conditions such as severe dry eye, diabetes, chronic exposure due to eyelid pathology, and ocular graft versus host disease following hematopoietic stem cell transplantation). In some embodiments, therapies using HGF variant polypeptides are used to treat, prevent and/or inhibit injury, previous ophthalmic surgery, infection (e.g., previous herpes infection or severe bacterial ulcer), or ocular disease (including underlying conditions such as severe dry eye, diabetes, chronic exposure due to eyelid pathology, and ocular graft versus host disease following hematopoietic stem cell transplantation).

For example, in adults, the HGF-Met pathway is involved in muscle regeneration following injury. Thus, the disclosed variants are useful for repairing muscle damage, including, for example, cardiac tissue regeneration following infarction.

The disclosed variants may be used, for example, to treat or prevent liver failure or disease caused by conditions including viral infection (such as infection by a hepatitis virus, e.g., HAV, HBV, or HCV) or other acute viral hepatitis, autoimmune chronic hepatitis, acute fatty liver of pregnancy, Budd-Chiari syndrome and venous occlusive disease, hyperthermia, hypoxia, malignant infiltration, Reye syndrome, sepsis, wilson's disease, and transplant rejection.

Typically, a therapeutically effective amount of a polypeptide variant will be in the range of about 0.1mg/kg to about 100mg/kg, optionally about 1mg/kg to 10 mg/kg. The amount administered will depend on variables such as the type and extent of the disease or indication to be treated, the overall health of the particular patient, the relative biological efficacy of the polypeptide variant delivered, the formulation of the polypeptide variant, the presence and type of excipients in the formulation, and the route of administration. In order to reach the desired blood or tissue level quickly, the initial dose administered may be increased beyond an upper level, or the initial dose may be less than optimal, and the daily dose may be gradually increased during the course of treatment, depending on the particular situation. Human doses can be optimized, for example, to 0.5mg/kg to 20mg/kg in a conventional phase I dose escalation study. The frequency of administration can vary depending on factors such as the route of administration, the dosage amount, and the disease condition being treated. Exemplary dosing frequencies are once daily, once weekly, and once every two weeks. A preferred route of administration is parenteral administration, such as intravenous infusion. Formulation of protein-based drugs is within the ordinary skill in the art. In some embodiments of the invention, polypeptide variants, e.g., protein-based polypeptides, are lyophilized and, upon administration, reconstituted in buffered saline.

The polypeptide variants may be administered alone or in combination with other pharmaceutically active ingredients. Other active ingredients, such as immunomodulators, may be administered with the polypeptide variant, or may be administered before or after the polypeptide variant.

Formulations comprising polypeptide variants for therapeutic use typically include the polypeptide variants in combination with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to buffers, carriers, and excipients which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. In this regard, pharmaceutically acceptable carriers are intended to include any and all buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like which are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.

The formulations may conveniently be presented in dosage unit form and may be prepared by any suitable method, including any method well known in the art of pharmacy. Remington's Pharmaceutical Sciences, 18 th edition (Mack Publishing Company, 1990).

In exemplary embodiments, the polypeptide variants are used for in vitro or in vivo diagnostic purposes, typically by directly or indirectly labeling the polypeptide variants with a detectable moiety. The detectable moiety may be any moiety capable of directly or indirectly producing a detectable signal. For example, the detectable moiety may be a radioisotope, such as³H、¹⁴C、³²P、³⁵S, or¹²⁵I; fluorescent or chemiluminescent compounds, such as fluorescein isothiocyanate, Cy5.5(GE Healthcare), AlexaDyes (Invitrogen),Infrared dye (A)Biosciences), rhodamine, or fluorescein; enzymes such as alkaline phosphatase, beta-galactosidase, or horseradish peroxidase; spin probes, such as spin labels; or colored particles such as latex or gold particles. It will be appreciated that polypeptide variants may be conjugated to a detectable moiety using a number of methods known in the art, for example as described in Hunter et al, (1962) Nature 144: 945; david et al, (1974) Biochemistry 13: 1014; pain et al, (1981) J.Immunol Meth 40: 219; and Nygren (1982) J.Histochem and Cytochem.30: 407. The markers may be detected, for example, visually or with the aid of a spectrophotometer or other detector or other suitable imaging system.

Polypeptide variants can be used in the field of available immunoassay technology. Exemplary immunoassays include, for example, sandwich immunoassays, competitive immunoassays, immunohistochemistry procedures.

In a sandwich immunoassay, two antibodies that bind the analyte or antigen of interest are used, e.g., one immobilized on a solid support and the other free in solution and labeled with a detectable moiety. When a sample containing an antigen is introduced into the system, the antigen binds to both the immobilized antibody and the labeled antibody, forming a "sandwich" immune complex on the surface of the support. The complex protein is detected by washing away unbound sample components and excess labeled antibody and measuring the amount of labeled antibody complexed with the protein on the surface of the support. Alternatively, free antibody in solution can be detected by a third antibody labeled with a detectable moiety that binds to the free antibody. A detailed review of immunoassay design, theory and protocol can be found in a number of documents, including Butt's eds, (1984) Practical Immunology, Marcel Dekker, New York; harlow et al (1988) Antibodies, A Laboratory apparatus, Cold Spring Harbor Laboratory; and Diamandis et al, (1996) Immunoassay, Academic Press, Boston.

Polypeptide of expected markerThe variants can be used as in vivo imaging agents, whereby the polypeptide variants can target the imaging agent to a particular tissue of interest in a recipient. Remotely detectable moieties for in vivo imaging include radioactive atoms⁹⁹mTc, a gamma emitter with a half-life of about 6 hours. Non-limiting examples of radionuclide diagnostic agents include, for example¹¹⁰In、¹¹¹In、¹⁷⁷Lu、¹⁸F、⁵²Fe、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁷Ga、⁶⁸Ga、⁸⁶Y、⁹⁰Y、⁸⁹Zr、⁹⁴mTc、⁹⁴Tc、⁹⁹mTc、¹²⁰I、¹²³I、¹²⁴I、¹²⁵I、¹³¹I、^154-158Gd、³²P、¹¹C、¹³N、¹⁵O、¹⁸⁶Re、¹⁸⁸Re、⁵¹Mn、⁵²mMn、⁵⁵Co、⁷²As、⁷⁵Br、⁷⁶Br、⁸²mRb、⁸³Sr or other gamma-, beta-or positron emitters.

Non-radioactive moieties also useful in vivo imaging include nitroxide spin labels as well as lanthanide and transition metal ions, all of which can induce proton relaxation in situ. In addition to imaging, the complexed radioactive moieties can also be used in standard radioimmunotherapy protocols to destroy target cells.

A wide variety of fluorescent labels are known in the art, including, but not limited to, fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine. Useful chemiluminescent labels may include luminol, isoluminol, an aromatic acridinium ester, imidazole, an acridinium salt, or an oxalate ester.

The disclosed polypeptide variants can also be labeled with fluorescent labels to allow in vivo detection. In some embodiments, the fluorescent label is cy5.5(GE Healthcare). In other embodiments, the fluorescent label is AlexaDye (Invitrogen). In some embodimentsIn the case, the fluorescent label isInfrared dye (A)Biosciences)。

Exemplary nucleotides for high dose radiation therapy include radioactive atoms⁹⁰Yt、¹³¹I and¹¹¹in. Coupling techniques known in the imaging arts can be used¹³¹I、¹¹¹In and⁹⁹a variant of a mTC-tagged polypeptide. Similarly, procedures for preparing and applying imaging agents and capturing and processing images are well known in the imaging arts and, therefore, are not discussed in detail herein. Similarly, methods for performing antibody-based immunotherapy are well known in the art. See, for example, U.S. patent No. 5,534,254.

Examples

Example 1: engineered hepatocyte growth factor dimer fragments for improved corneal epithelial wound healing in vitro

Background

Hepatocyte Growth Factor (HGF) is a naturally occurring mitogen that plays a key role in corneal wound healing. NK1 is an engineered fragment of HGF that weakly activates the same c-Met receptor. NK1 was previously engineered using directed evolution to achieve improved stability and recombinant expression yield.

Disulfide-linked NK1 homodimers were created by the introduction of an N-terminal cysteine residue. Compared with wild type NK1, the engineered NK1 covalent dimer showed an increase of approximately one order of magnitude in agonistic activity, approaching that of full-length HGF. The CD spectrum shows that the dimer has improved thermostability compared to wild-type NK 1.

Purpose(s) to

Hepatocyte Growth Factor (HGF) is a naturally occurring mitogen that plays a key role in corneal wound healing. This example describes the wound healing properties of engineered HGF dimer fragment eNK1 dimer on human primary Corneal Epithelial Cells (CECs) by migration and proliferation assays. It is hypothesized that CEC migration and proliferation were improved using eNK1, and eNK1 performed similarly to recombinant hgf (rhgf).

Method

NK1 is a fragment of HGF that weakly activates the c-Met receptor. NK1 was previously engineered using directed evolution to achieve improved stability and recombinant expression yield. Disulfide-linked NK1 homodimers were created by the introduction of an N-terminal cysteine residue. Migration and proliferation assays were performed on immortalized CECs and primary CECs, respectively. Migration of immortalized CECs was assessed by scratch assay with 100ng/mL of eNK1 and rHGF treatment. Wound closure was monitored under an inverted microscope at 6 and 12 hours. Proliferation and metabolic activity of primary CECs were assessed after 48 hours. Cells were first starved in growth factor-free medium and then treated with 100ng/mL of eNK1 or rHGF. Cell proliferation and metabolic activity were quantified using the Click-iT EdU and MTT assays, respectively.

Migration and proliferation assays were performed on immortalized CECs and primary CECs, respectively. Migration of immortalized CECs was assessed by scratch assay with 100ng/mL of eNK1 and rHGF treatment. Wound closure was monitored under an inverted microscope at 6 and 12 hours. Proliferation and metabolic activity of primary CECs were assessed after 48 hours of treatment. Cells were first starved in growth factor-free medium and then treated with 100ng/mL of eNK1 or rHGF. Cell proliferation and metabolic activity were quantified using the Click-iT EdU and MTT assays, respectively.

Results

CEC treated with eNK1 and HGF had increased wound closure rate

CECs treated with both eNK1 and rHGF increased wound closure rates compared to untreated cells. The scoring assay showed that CECs treated with both eNK1 and rHGF achieved significantly greater wound closure (p <0.01) compared to the negative control. See fig. 3.

eNK1 and HGF treated cells have increased cell proliferation

The EdU assay showed that CECs treated with eNK1 and rHGF for 48 hours were significantly more EdU positive than untreated cells. Red cells represent a fluorescent label for newly synthesized DNA, indicating increased levels of DNA synthesis and thus increased levels of proliferation in cells treated with rHGF and eNK1 (p < 0.05). See fig. 4.

MTT proliferation assays showed that CECs treated with eNK1 and rHGF for 48 hours had increased metabolic activity (p <0.05) compared to untreated cells. See fig. 5.

Compared with wild type NK1, the engineered NK1 covalent dimer showed an increase of approximately one order of magnitude in agonistic activity, approaching that of full-length HGF. The CD spectrum shows that the dimer has improved thermostability compared to wild-type NK 1. The scoring assay showed that CECs treated with both eNK1 and rHGF achieved significantly greater wound closure (p <0.01) compared to the negative control. EdU assay showed CECs treated with eNK1 and rHGF were significantly more EdU positive than untreated cells, indicating increased levels of DNA synthesis and thus increased levels of proliferation (p < 0.05). MTT assays showed CECs treated with eNK1 and rHGF had increased metabolic activity compared to untreated cells (p < 0.05).

Conclusion

The data provided in this example supports our following assumptions: eNK1 is a stable protein that affects the migration, proliferation and metabolic activity of corneal epithelial cells to levels similar to full length recombinant HGF. The results indicate eNK1 improved wound healing of corneal epithelium and potential as a therapeutic alternative to recombinant HGF.

Further in vitro and in vivo studies will focus on eNK1 at different concentrations and in vehicles with different biomechanical properties to enhance corneal wound healing and to prevent scarring and angiogenesis.

Example 2: protein engineering of NK1

1.1 protein engineering of NK1 by yeast surface display. Yeast surface display is a powerful directed evolution technique that has been used to engineer proteins for enhanced binding affinity, proper folding, and improved stability. Tong (Chinese character of 'tong')A combinatorial library of NK1 protein was displayed on the surface of the yeast strain Saccharomyces cerevisiae by genetic fusion with the yeast conjugated lectin protein Aga2 p. Aga2p was bound to Aga1p by a disulfide bond, which was covalently linked to the yeast cell wall. Unlike most yeast display studies, the constructs used herein tethered the displayed NK1 protein to the N-terminus of Aga2p (figure 2 from U.S. patent No.9,556,248). For this ligand-receptor system, this orientation was found to reduce the spatial limitations of receptor and antibody labeling described below. The NK1 protein was flanked by an N-terminal Hemagglutinin (HA) and a C-terminal C-myc epitope tag, which was used to confirm the expression of the construct on the yeast cell surface and to quantify the surface expression level. Use of flexibility (Gly) at the C-terminus of displayed NK1 protein₄Ser)₃The linker projects the protein away from the yeast cell surface to further minimize spatial limitations.

Is generally created 10⁷To 10⁸A library of individual transformants for protein engineering studies in which each yeast cell displays thousands of identical copies of a particular NK1 mutant on its surface. High throughput screening of tens of millions of yeast-displayed NK1 mutants using Fluorescence Activated Cell Sorting (FACS) allows the isolation of protein variants with the desired properties, in this case enhanced Met receptor binding affinity and/or enhanced expression. For this purpose, the yeast-displayed NK1 library was stained with both fluorescently labeled Met-Fc fusion protein and primary and secondary antibodies against the HA epitope tag (figure 2B from us patent No.9,556,248). The use of multi-color flow cytometry enables simultaneous and independent monitoring of both relative surface expression levels and Met binding by detecting phycoerythrin and Alexa-488 fluorescence, respectively. Yeast cells were isolated that bound the highest level of Met and had the highest expression level of NK 1. Previously, it has been shown that there is a strong correlation between expression levels on the surface of yeast cells and thermostability and soluble expression yield. The sorted yeasts are propagated in culture and the screening process is repeated several times to obtain an enriched yeast population consisting of a small number of unique clones.

1.2 overview: yeast surface display was used to guide the evolution of NK1 to achieve high affinity and stability. The NK1 fragment was engineered to achieve 1) enhanced thermostability and 2) high binding affinity for Met. The first round of directed evolution consisted mainly of the evolution NK1 to perform functional expression on the yeast cell surface and achieve modest improvement in Met binding affinity. The pooled products were further mutagenized and subjected to a second round of directed evolution in which they were screened independently for increased Met binding affinity or enhanced stability. A third round of directed evolution was then performed by performing DNA shuffling on pooled products from the second round, followed by simultaneous screening for increased Met binding affinity and enhanced stability (fig. 3).

1.3 wild type NK1 was not functionally expressed on the yeast cell surface. HGF exists in two major isoforms, isoform 1 (I1: Genbank accession NP-000592) and isoform 3 (I3: Genbank accession NP-001010932; SEQ ID NO: 10).

HGF I1 and I3 were identical in sequence, except that there were 5 amino acid deletions in the first Kringle domain (K1) of I3. Yeast display plasmid pTMY-HA was used to express NK 1I 1 or NK 1I 3 on the yeast cell surface as a genetic fusion with yeast cell wall protein Aga2p (fig. 2). Similar results were found for both NK 1I 1 and NK 1I 3. Yeast displayed NK 1I 1 was stained for relative expression (detected by HA-tagged antibodies) and binding to 20nM or 200nM Met-Fc labeled with Alexa 488 (R & D Systems). Since heparin is required for the wild-type NK1-Met interaction, the experiment was performed in the presence (top of fig. 4A from U.S. patent No.9,556,248) and absence (bottom of fig. 4A) of 2 μ M heparin (Lovenox, Sanofi-Aventis). Flow cytometry was used to detect yeast expressing NK 1I 1 on the surface of yeast cells. Only low levels of binding to soluble Met-Fc were observed (figure 4, x and y axes). The binding level after heating yeast-displayed NK1 to 70 ℃ is shown (figure 4B from us patent No.9,556,248). As shown below, soluble NK 1I 1 produced by the yeast Pichia pastoris (Pichia pastoris) completely unfolded at 60 ℃ (FIG. 4 from U.S. Pat. No.9,556,248). Overall, the data indicate that yeast displayed wild-type NK1 is not functionally expressed on the yeast cell surface.

1.4 engineering NK1 using yeast surface display to achieve improved affinity and stability. Three separate rounds of directed evolution were used to evolve NK1 to achieve improvements in stability and Met binding affinity compared to wild-type NK 1. Since NK1 is not functionally expressed on the yeast cell surface, the first round of directed evolution mainly consisted of screening yeast-displayed NK1 mutants to isolate clones that bound to the Met receptor. To achieve this goal, approximately 3X 10 was generated by error-prone PCR using the nucleotide analogs 8-oxo-dGTP and dPTP (TriLink Biotechnologies)⁷Library of individual NK1 mutants. Since neither NK 1I 1 nor NK 1I 3 were functionally expressed on the yeast cell surface, it is not clear which isoform is most suitable for affinity maturation by directed evolution. Thus, equal amounts of NK 1I 1 and NK 1I 3 were used as starting templates to generate a combinatorial NK1 mutant library based on both I1 and I3. Sequencing of random clones from the yeast-displayed library confirmed an equal representation of NK 1I 1 and NK 1I 3.

Yeasts prefer to grow at 30 ℃, however, they tend to show improved expression of more complex proteins at 20 ℃. Thus, two rounds of library sorting were performed after induction of protein expression on the yeast cell surface at 20 ℃ to achieve improved NK1 mutant folding, and FACS was used to isolate yeast cells that exhibited detectable binding to 200nM Alexa-488 labeled Met-Fc (Met-Fc a488) (figure 5A from U.S. patent No.9,556,248). Subsequent library classification was performed in parallel using either 20 ℃ or 30 ℃ induction temperature in order to screen for mutants with improved stability using an expression temperature of 30 ℃. After five rounds of sorting (5 rounds using an expression temperature of 20 ℃ C., or 2 rounds using 20 ℃ C., followed by 3 rounds using an expression temperature of 30 ℃ C.) with each strategy, the library clearly contained members that bound to 200nM Met-Fc.

For the second round of directed evolution, pooled mutants from the final species from the first round of directed evolution were randomly mutated by error-prone PCR to generate approximately 8 × 10⁷A library of individual unique mutants. Two previous rounds of sorting of the library were performed using an expression temperature of 20 ℃ to first recover the mutants that bound to soluble Met-Fc a 488. For subsequent rounds, the increase in expression (i.e., folding stability), which has been shown to correlate with increased thermostability, or the increase in binding affinity for sorted Met (figure 5B from U.S. patent No.9,556,248) was sorted in parallel. Expression at elevated temperatures (37 ℃) was used to confer sorting stringency to achieve increased stability, while increased binding to ever decreasing concentrations of soluble Met-Fc a488 was used to achieve affinity sorting stringency.

Finally, the third round of directed evolution consisted of DNA shuffling of the final pool of stability and affinity enhanced mutants from the second round of directed evolution to generate about 2 × 10⁷Third generation libraries of individual unique transformants. The library was simultaneously screened for enhanced stability (by high cell surface expression levels upon induction at 37 ℃) and enhanced affinity (by increased binding to substantially decreasing concentrations of Met-Fc a 488). The first, second and third rounds of sorting used 40nM, 20nM and 2nM Met-Fc A488, respectively. After three rounds of sorting, the resulting library of mutants was well expressed at 37 ℃ and strongly bound to 2nM Met-Fc A488 (FIG. 6, middle from U.S. Pat. No.9,556,248). Subsequent sorting was performed by: labeling with 2nM Met-Fc A488 followed by recombinant HGF (R) in the presence of excess unlabeled competitor (in this case&D system)) is performed. Clones that retained Met binding after 24 hours in the presence of excess HGF competitor were isolated by FACS. This process was repeated until after a2 day unbinding step in the presence of excess HGF as Met-Fc competitor, the NK1 mutant pool retained binding to Met-Fc a488 (fig. 6, right).

A bank of NK1 variants was identified, wherein the variants were efficiently expressed on the yeast cell surface at elevated temperatures and maintained persistent binding to 2nM soluble Met even after a2 day unbinding step in the presence of excess HGF competitor (figure 6 from U.S. patent No.9,556,248).

1.5 sequence analysis of NK1 mutant with enhanced affinity and stability. In parallel with performing round 3 directed evolution, promising mutants from round 2 began to be characterized. The 8 random mutants from each of the last two sorting runs were sequenced for each sorting strategy (20 ℃ affinity sorting strategy and 37 ℃ stability sorting strategy). Interestingly, all 32 clones sequenced were based on NK 1I 1, even though sequencing of the initial library showed relatively equal proportions of NK1 isoform 1 and isoform 3. In addition, a number of favorable or consistent mutations are evident. 10 mutations were repeated from randomly sequenced clones in the library sort products, and 8 of these mutations appeared in more than half of the randomly selected clones. These dominant mutations are highlighted in bold in table 1. Because of the wide variety of mutations, no single clone contained all 8 of these mutations. However, one clone contained five of the eight most common mutations (K62E, N127D, K137R, K170E, N193D; this clone was designated M2.1). The remaining three mutations (Q95R, K132N, Q173R) were added to the background of this clone to generate the NK1 mutant, designated M2.2. Further sequence analysis of these mutations highlights a number of interesting observations, which are discussed further below.

The sorted products from both strategies did not produce many identical clones, but showed significant overlap in the consensus sequence. The negative correlation between I125T and N127D observed in the M2 (second round of directed evolution) product persists in the M3 (third round of directed evolution) product. Of the 30 sequenced clones, 25 contained the N127D mutation, none of which also contained the I125T mutation. However, each of the five clones that did not contain N127D contained the I125T mutation. The K62E/V64A and I130V/K132N consensus mutation occurred with only 2 amino acid intervals.

All eight consensus mutations from the M2 product were present in the M3 product (recall that M2.2 ═ K62E, Q95R, N127D, K132N, K137R, K170E, Q173R, N193D). Over 50% of the M3 products showed five additional consensus mutations: V64A, N77S, I130V, S154A and F190Y.

Table 7: sequence substitutions present in certain variants

Example 3: production of NK1

2.1 soluble production of wild type NK1 and NK1 mutants in the yeast strain Pichia pastoris (P.pastoris). Briefly, DNA encoding wild type NK1, M2.1, or M2.2 containing an N-terminal FLAG epitope tag (DYKDDDDK) and a C-terminal hexahistidine tag was cloned into the secretory plasmid pPIC 9K. Constructs were transformed into Pichia pastoris and selection for growth on YPD agar plates containing 4mg/mL geneticin and screening for NK1 expression by Western blotting of culture supernatants. Figure 7A (from us patent No.9,556,248) shows that M2.1 and M2.2 are well expressed at 30 ℃, whereas wild type NK1 is expressed at a much lower level. This data is consistent with previous studies reporting the use of yeast surface display engineering to achieve enhanced protein stability, as well as conferring increased levels of recombinant expression. However, lowering the expression temperature to 20 ℃ enabled efficient expression of wild-type NK1 (data not shown). NK1 and mutant expression was amplified to 0.5L in shake flask culture and purified using immobilized nickel affinity chromatography followed by Superdex^TMGel filtration was performed on a 75 column (GE Healthcare). Without any optimization, several milligrams of mutants M2.1 and M2.2 were obtained from one 0.5L flask, indicating that higher yields can be obtained by changing the induction conditions or by fermentation.

2.2 mutants M2.1 and M2.2 showed higher thermostability than wild type NK 1. To test the thermostability, M2.1 and M2.2 were expressed on the yeast cell surface, heated to different temperatures, and the retention of binding to fluorescently labeled Met-Fc was measured by flow cytometry (from us patent No.9,556,248, fig. 8A). T of NK1 mutants M2.1 and M2.2 on the surface of Yeast_mThe values were 61.0. + -. 1.4 ℃ and 61.4. + -. 0.7 ℃ respectively. Since the yeast-displayed wild-type NK1 was not functionally expressed on the yeast cell surface, the stability of the yeast-displayed wild-type NK1 could not be monitored.

To test the stability of soluble proteins, secondary structure unfolding of purified soluble mutants was monitored on a Jasco J-815CD spectrometer using Circular Dichroism (CD). CD scanning of the mutant protein identified a peak at 208nm, which was largely due to the beta sheet structural element. The CD scans of M2.1 and M2.2 were similar to wild-type NK1, indicating that the mutein contains the same overall secondary structural elements as wild-type NK1 (from figure 8B of U.S. patent No.9,556,248). The CD spectrum of wild type NK1 at 80 ℃ was similar to that of the random coil, indicating the ability to monitor secondary structural element unfolding using circular dichroism (fig. 8B). Using this information, the unfolding of this secondary structure can be monitored by variable temperature CD scanning (fig. 8C). In each of these assays, wild-type NK1 (T)_mAs compared to 50.9 ± 0.2 ℃), M2.1 and M2.2 exhibited higher thermal stability (63.6 ± 0.3 ℃ and 67.8 ± 0.2 ℃, respectively). To further confirm these results, the melting of the local maximum at 236nm for M2.1 was monitored as a function of the melting of the random coil. The same T was observed for melting at 208nm_m. Table 8 shows the thermostability (T) of wild type and mutant NK1 proteins as determined by CD temperature melting_m) To summarize (a).

Table 8.

	Tm + standard deviation (. degree.C.)
		NK1	50.7+0.2
NK1 N127A	47.9+0.7
		M2.1	63.9+0.5
M2.2	69.0+1
		M2.2 D127N	65.5+0.5
M2.2 D127A	63.7+0.1
		M2.2 D127K	62.5+0.1
M2.2 D127R	62.3+0.5

2.3 Effect of salt concentration on protein stability. To maintain its structural integrity, it has been observed that wild-type NK1 must be kept in a buffer containing a high salt concentration (>200-300mM NaCl). As further evidence of this requirement, wild-type NK1 showed a broad delayed elution profile on size exclusion chromatography using a buffer containing a moderate salt concentration (137mM) (fig. 9 and inset from U.S. patent No.9,556,248), indicating unfolding and/or non-specific binding to the column under these conditions. In contrast, under similar moderate salt conditions, M2.1 and M2.2 eluted as a single sharp peak on size exclusion chromatography (figure 9 from U.S. patent No.9,556,248).

Example 4: characterization of NK1

3.1NK1 homodimerization interface point mutation residue N127 located in the N domain and K1 domain connecting the joint region (figure 1). The side chains of the asparagine residue form two hydrogen bonds. Among the variants isolated from the library, the N127D variant was frequently observed. (tables 2 and 3). To explore the effect of the N127D mutation in M2.2 on biological activity, a series of point mutants were generated at this position. Alanine residues convert wild-type NK1 from agonist to antagonist by disrupting the stabilizing interaction of the NK1 homodimer. The effect of mutation at this position to lysine or arginine was tested. These substitutions introduce steric and electrostatic barriers through bulky charged side chains.

In addition, point mutant D127N was analyzed; this will reduce this position back to the wild-type asparagine residue. In the context of M2.2 comprising the N127D mutation, these mutations are referred to as D127A, D127K, D127R and D127N. Importantly, each of these mutants retained the high thermal stability associated with M2.2 (table 5).

3.2 characterization of NK1 mutant as Met receptor agonist or antagonist. NK1 mutants were evaluated in MDCK cell dispersion (cell scatter) and uPA assays, two assays widely used to study Met receptor activation in mammalian cells. For MDCK cell dispersion assays, 1500 cells/well were seeded into 100 μ L of complete growth medium in 96-well plates and 5% CO at 37 ℃₂And (4) incubating. After 24 hours, the medium was aspirated and replaced with medium containing HGF or NK1 protein at concentrations of 0.1nM or 100nM, respectively. In some of the experiments that were carried out,heparin (Sanofi-Aventis) was used at a concentration of 2. mu.M or at a 2:1 molar ratio of heparin: NK 1. After 24 hours, the cells were fixed and stained with 0.5% crystal violet in 50% ethanol solution for 10 minutes at room temperature, washed with water, and air-dried, and photographed. MDCK dispersion inhibition assays were performed in a similar manner except that cells were incubated with 250nM NK1 mutant for 30 minutes, followed by addition of HGF to a final concentration of 0.1 nM.

For the MDCK uPA assay,4000 cells/well were seeded into 100. mu.L of complete growth medium in 96-well plates and incubated at 37 ℃ with 5% CO₂And (4) incubating. After 24 hours, the medium was aspirated and replaced with medium containing HGF or NK1 at concentrations of 1nM or 100nM, respectively. After 24 hours, the cells were washed twice with 200 μ L of phenol red-free DMEM and incubated with 200 μ L of reaction buffer containing 50% (v/v) of 0.05 units/mL plasminogen (Roche Applied Science), 40% (v/v) of 50mM Tris pH 8.0, 10% (v/v) and 3mM chromozym PL (Roche Applied Science) in 100mM glycine pH 3.5. The plates were incubated at 37 ℃ with 5% CO₂Incubate for 4 hours, then use the Infinite M1000 enzyme-linked immunosorbent assay (Tecan Group Ltd.) measurement at 405nm absorbance.

Mutants M2.2D127A, D127K and D127R did not induce Met activation as measured by the spread of MDCK cells (fig. 10 and 11A from U.S. patent No.9,556,248) or uPA activation in MDCK cells (fig. 11B). The unmodified M2.2 variant containing the N127D mutation exhibited weak activity (from figure 11A of U.S. patent No.9,556,248) or no agonistic activity (from figures 10 and 11B of U.S. patent No.9,556,248).

In contrast, restoration of position 127 to the wild-type asparagine residue (M2.2D 127N) resulted in agonistic activity in both MDCK dispersal (from fig. 10 and 11A of U.S. patent No.9,556,248) and uPA assay (from fig. 11B of U.S. patent No.9,556,248). The activity of M2.2D 127N was similar to that of wild-type NK1 and all showed enhanced activity in the presence of soluble heparin (top vs bottom of figure 11C from us patent No.9,556,248). In contrast, none of M2.2D127A, D127K, and D127R exhibited agonistic activity in these assays in the presence or absence of heparin (figures 10 and 11A-11C from U.S. patent No.9,556,248).

These mutants were tested for their ability to inhibit HGF-induced Met activation. As a control, M2.2D 127N did not inhibit HGF-induced activity, thereby providing further evidence of its function as a Met receptor agonist (figure 12 from U.S. patent No.9,556,248). M2.2 mutants D127A, D127K and D127R showed weak or minimal inhibition of HGF-induced MDCK dispersion in the absence of soluble heparin (from the top of fig. 12 of U.S. patent No.9,556,248).

In contrast, strong antagonistic activity was observed with the addition of 2 μ M heparin (bottom of fig. 12). Pre-formulating NK1 mutant with a 2:1 molar ratio of heparin to NK1 was sufficient to confer this antagonistic activity and eliminated the need to add excess heparin to improve the antagonistic activity (figure 13 from us patent No.9,556,248). Unmodified M2.2 (M2.2N 127D) showed only weak antagonistic activity with 2:1 molar ratio of heparin (from figure 13 of us patent No.9,556,248), supporting the utility of rationally designed point mutations. The antagonistic activity of M2.2D 127K was similar to that previously reported for the antagonist NK 1N 127A (from figure 13 of U.S. patent No.9,556,248). However, the M2.2D 127A/K/and R mutant has significantly improved stability and expression compared to NK 1N 127A, i.e. lower salt dependent stability, increased T of about 15 ℃_mAnd about 40-fold improved recombinant expression yield, all of which are attractive properties.

4.1 Biochemical and biological characterization of recombinant Aras-4. The clones were selected for further study based on the sequence distribution, yeast surface expression level and Met-Fc binding of five clones from the third round of directed evolution. These clones were designated Aras-1, Aras-2, Aras-3, Aras-4 and Aras-5 (FIG. 14 from U.S. Pat. No.9,556,248). Each of these clones, except for Aras-1, was found to be well expressed in the yeast Pichia pastoris.

Aras-4 was chosen for further characterization. E.g. by CD temperature melting (T)_mDetermined at 64.9 ± 1.2 ℃), it exhibits high thermal stability. Aras-4 does not activate cellular Met when added to MDCK cell cultures and effectively inhibits HGF-induced Met activation at a concentration approximately five-fold lower than M2.2D127A or the wild-type NK 1-based antagonist NK 1N 127A (figure 15 from U.S. patent No.9,556,248).

4.2 introduction of disulfide bonds to form covalently bound dimers. A free cysteine residue was introduced into the N-terminus of M2.2D 127N, which resulted in the formation of monomeric and dimeric species upon recombinant expression. Cystine-linked dimeric protein (designated cdD127N) was purified from monomers using size exclusion chromatography. SDS-PAGE analysis of cdD127N under reducing and non-reducing conditions confirmed that dimers were formed by covalent disulfide bonds. (FIG. 16 from U.S. Pat. No.9,556,248). Cystine-linked dimer M2.2D 127K (designated cdD127K) and Aras-4 (designated cdAras-4) polypeptides were also produced.

4.3cdD127N, cdD127K and cdAras-4. cdD127N and cdD127K showed agonistic activity at orders of magnitude lower concentrations than M2.2D 127N monomer, which has similar agonistic activity as wild-type NK1 (figure 17 from us patent No.9,556,248). cdD127K was surprising in that the parent monomer M2.2D 127K was an antagonist. Also surprising is the result of cdAras-4, where the covalent bond converts the antagonist Aras-4 to an agonist. The observed level of agonistic activity is close to full-length HGF, however cdD127N, cdD127K and cdAras-4 have significantly improved stability relative to full-length HGF and can be recombinantly expressed in yeast.

4.4 Only the N-terminal cysteine directly mediates homodimerization. Based on the crystal structure of NK1 homodimer, it was recognized that position 127 is very close on the adjacent promoter. This suggests the possibility of forming covalently linked homodimers by placing a cysteine residue at this position. To test this possibility, variant Aras-4 polypeptides were generated in which residue D127 was substituted with Cys. As shown in fig. 18 (from U.S. patent No.9,556,248), the resulting polypeptide was essentially unable to produce dimers either spontaneously or after phenanthroline-copper sulfate treatment.

In addition to covalent attachment by adding a free cysteine at the N-terminus of NK1 and its variants, other positions and linkers were tested. (FIG. 19 from U.S. Pat. No.9,556,248). Free cysteine or a combination of free cysteine and cysteine tags (Backer et al (2006) nat. Med.13(4):504- & 509) are attached to the N-terminus or C-terminus of the Aras-4 variant. Upon recombinant expression in yeast, only the free cysteine at the N-terminus produces the dimeric protein.

5.0 heparin-containing sea foodPreparation of HGF variant polypeptide of alginate micro-pill. Calcium alginate pellets can provide a stable platform for HGF due to enhanced activity retention and extended storage time, and thus can be used as a device for controlled HGF variant release. Heparin-sepharose beads (Pharmacia LKB) can be sterilized under UV light for 30 minutes and then mixed with filter-sterilized sodium alginate. The mixed slurry can then be dropped through a needle into the CaCl-containing vessel₂(1.5% w/v) in a beaker of sclerosant solution. The beads can be formed immediately. By encapsulating in CaCl₂Cross-linked capsule envelopes can be obtained by incubation in solution for 5 minutes with gentle mixing and then 10 minutes without mixing. The beads formed can be washed with sterile water and stored at 4 ℃ in 0.9% NaCl-1mmol/L CaCl₂In (1). Can pass through 10 capsules at 0.9% NaCl-1mmol/L CaCl₂HGF loading was performed with 0.05% gelatin and 12.5 μ g (for 10 μ g dose) or 125 μ g (for 100 μ g dose) or HGF variants incubated at 4 ℃ for 16 hours with slow stirring. The final product can be sterilized under uv light for 30 minutes.